Formulation of Anti-CD40l antibody and preparation method for formulation

Abstract

The present invention relates to the field of pharmaceutical formulations, and provides a formulation of an anti-CD40L antibody and a preparation method for the formulation. More specifically, the present invention provides a formulation of a recombinant humanized anti-cluster of differentiation antigen CD40 ligand (CD40L) antibody and a preparation method for the formulation, and also relates to a use of the formulation in treating diseases or conditions related to inappropriate activation of a CD40L / CD40 mediated pathway.

Description

Formulation and preparation method of anti-CD40L antibody

[0001] This application claims the benefit of Chinese Invention Application No. 202411510409.8, filed on October 28, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical formulations. More specifically, this invention relates to formulations of recombinant humanized anti-differentiation cluster antigen CD40 ligand (CD40L) antibodies and methods for preparing the same, and also to the use of said formulations for treating diseases or conditions related to inappropriate activation of the CD40L / CD40-mediated pathway.

[0003] Background of the Invention

[0004] CD40L, also known as CD154, gp39, TNF-associated activating protein (TRAP), 5c8 antigen, or T-BAM, is a 39 kDa type II transmembrane protein. CD40L belongs to the tumor necrosis factor (TNF) superfamily and exists on the cell membrane as a homotrimer. CD40L is primarily expressed on activated CD4+. + CD40L is present on the surface of T cells and also on other types of immune and non-immune cells, such as epithelial cells, monocytes, dendritic cells, fibroblasts, smooth muscle cells, endothelial cells, and platelets. CD40L interacts with CD40 expressed on the surface of B cells and plays an important role in various immune responses and inflammatory processes. For example, it mediates humoral immune responses and the release of inflammatory factors; it acts as a co-stimulatory molecule in the interaction between T cells and antigen-presenting cells (APCs) and B cells; and it plays a crucial role in germinal center (GC) formation and antibody class switching. Therefore, the CD40L / CD40 signaling pathway plays an important pathogenic role in various B cell-related diseases, such as autoimmune diseases, neurodegenerative diseases, graft-versus-host disease, tumors, and atherosclerosis.

[0005] Currently, several antibodies or protein drugs targeting CD40L have shown positive clinical efficacy. Early antibody Ruplizumab (CN101072587B) showed good therapeutic effects in diseases such as systemic lupus erythematosus and lupus nephritis, but its development was discontinued due to serious safety events such as thrombosis (Tatsuo Kawai et al., Thromboembolic complications after treatment with monoclonal antibody against CD40ligand, Nat. Med. 6(2)(2000) 114). Subsequent studies have shown that thrombosis mainly occurs because free CD40L forms an immune complex with the antibody, which then interacts with C1q and FcgR, especially with FcgRIIa expressed on platelets, inducing platelet activation and aggregation, leading to thrombosis. Therefore, completely eliminating the effector function of the Fc region is crucial for the safety of anti-CD40L antibodies.

[0006] Antibodies targeting CD40L that subsequently entered clinical trials all modified the Fc region, primarily through two technical approaches: one is to weaken the effector function of the Fc region through mutation; the other is to completely remove the Fc region while using other techniques to extend the molecule's half-life. However, both approaches have certain problems. In the first approach, taking frexalimab (AU2016294417B2) as an example, both used C220S / C226S / C229S / P238S and E269R / K322A mutations to weaken the effector function of the Fc region, but subsequent studies showed that these mutations could not completely remove the effector function of the Fc region, thus posing certain safety risks. The second approach, exemplified by Dapirolizumab pegol (US8293237B2) and Dazodalibep (CN101679521B), involves retaining only the monovalent Fab of the CD40L antibody and extending its half-life through cross-linking with PEG. However, this monovalent molecular form weakens the molecule's biological activity, and PEG cross-linking also increases the difficulty of molecular CMC (Chemistry, Manufacturing and Controls). Dazodalibep uses a non-traditional antibody form of the Tn3 backbone protein as a molecule that targets and inhibits CD40L, and extends the molecule's half-life by fusing with albumin. However, clinical studies have shown that this form of molecule has high immunogenicity, and therefore also poses certain safety risks.

[0007] The applicant disclosed novel CD40L antibodies in its previously filed international application PCT / CN2024 / 090122. These antibodies utilize the molecular form of IgG, and through Fc mutation technology, the molecule completely removes the binding ability of the Fc region to all FcγRs while retaining affinity for FcRn. Therefore, the antibodies disclosed in this application, while reducing the immunogenicity and safety risks of the molecule, retain the favorable pharmacokinetic characteristics of antibody molecules, making them highly promising antibody drugs. The entire contents of international application PCT / CN2024 / 090122 are incorporated herein by reference as if their entire contents were listed in detail herein.

[0008] Drug stability is a crucial indicator of drug efficacy and safety. Obtaining a suitable formulation is key to maintaining the efficacy and safety of a drug throughout its shelf life. However, protein formulations are prone to degradation and aggregation during formulation, storage, and use, which can affect the safety and efficacy of the drug product. Furthermore, protein solubility can be affected by various factors, such as pH and excipients, often making it difficult to formulate liquid formulations due to insufficient solubility. The sensitivity of proteins necessitates mild conditions during formulation to avoid damaging their structure and function. Due to the complexity of proteins themselves and their degradation pathways, it is currently impossible to predict the formulation conditions required for optimizing antibody stability, especially considering that different antibodies typically have different CDR sequences, and these sequence differences lead to different stability properties in solution. Therefore, based on the stringent requirements for the safety and efficacy of human antibodies, it is necessary to optimize individualized formulations for antibodies. The antibody disclosed in international application PCT / CN2024 / 090122 is prone to deamidation, leading to drastic changes in the acid-base composition of the charge variant, which further increases the difficulty of formulating it into a drug product.

[0009] The present invention aims to provide a stable pharmaceutical formulation of the anti-CD40L antibody or an antibody with a similar structure disclosed in international application PCT / CN2024 / 090122, so that it can be used clinically.

[0010] Invention Overview

[0011] In a first aspect, the present invention provides a pharmaceutical formulation comprising an anti-CD40L antibody or an antigen-binding fragment thereof, a pH adjuster, a protein protectant, and a surfactant, wherein the formulation is a lyophilized formulation or a liquid formulation, and the liquid formulation has a pH value of 5.0-6.0.

[0012] In a second aspect, the present invention provides a pillbox containing the pharmaceutical preparation described in the first aspect, and instructions for using the preparation to prevent or treat an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway.

[0013] In a third aspect, the present invention provides a pre-filled syringe containing the pharmaceutical preparation described in the first aspect.

[0014] In a fourth aspect, the present invention provides a method for preparing the pharmaceutical formulation described in the first aspect, the method comprising the following steps:

[0015] (1) Dissolve the pH adjuster and protein protectant in an injectable solvent, add the anti-CD40L antibody or its antigen-binding fragment, and optionally adjust the pH value to pH 5.0-6.0;

[0016] (2) Add a surfactant to obtain the liquid formulation;

[0017] Optionally, (3) the liquid formulation obtained in step (2) is freeze-dried to obtain the freeze-dried formulation.

[0018] In a fifth aspect, the present invention provides the use of the pharmaceutical preparations described in the first aspect for the prevention or treatment of diseases or conditions related to inappropriate activation of the CD40L / CD40-mediated pathway.

[0019] In a seventh aspect, the present invention provides a method for preventing or treating an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway, the method comprising injecting the individual with an effective amount of the pharmaceutical preparation described in the first aspect.

[0020] Brief description of the attached figures

[0021] Figure 1 shows the trend of the results of the pre-formulation study of Example 1, where (A) is the trend of protein content (UV method); (B) is the trend of purity (SEC-HPLC method); (C) is the trend of purity (non-reducing CE-SDS method); and (D) is the trend of charge variant-principal component (iCIEF method).

[0022] Figure 2 shows the change trends of charge variants of liquid formulations F1 to F6 of Example 2 after being placed at 40°C for 2 weeks. The upper figure shows the change trend of the main component, and the lower figure shows the change trend of the acidic component.

[0023] Figure 3 shows the stability test results of the liquid and lyophilized formulations of Examples F7 to F9 in Example 3 after being placed at 40°C for 2 weeks. Among them, (A) is the trend graph of antibody A purity (SEC-HPLC method) in the liquid formulation; (B) is the trend graph of antibody A purity (non-reducing CE-SDS method) in the liquid formulation; (C) is the trend graph of charge variant-principal component (iCIEF method) in the liquid formulation; (D) is the trend graph of antibody A purity (SEC-HPLC method) in the lyophilized formulation; (E) is the trend graph of antibody A purity (non-reducing CE-SDS method) in the lyophilized formulation; and (F) is the trend graph of charge variant-principal component (iCIEF method) in the lyophilized formulation.

[0024] Figure 4 is a schematic diagram of the structure of antibody A.

[0025] Figure 5 shows the amino acid sequence of antibody A. The variable region sequence is indicated in blue (amino acids 1-116 of the heavy chain and 1-111 of the light chain), the constant region sequence is indicated in black (the part outside the variable region), the complementarity-determining region (CDR) is underlined, the glycosylation site (N296) is indicated in bold black, intrachain disulfide bond pairing sites are connected by lines, and interchain disulfide bond pairing information is as follows: Heavy chain C 219 -Light chain C 218 Heavy chain C 225 -Heavy chain C 225 Heavy chain C 228 -Heavy chain C 228 .

[0026] Figures 6, 7, and 8 show data for antibody A in an experimental autoimmune encephalomyelitis (EAE) mouse model.

[0027] Figure 9 shows that antibody A inhibits the production of antihemocyanin antibody in mice.

[0028] Invention Details

[0029] definition

[0030] For the purposes of this invention, the terms used herein have the meanings defined below. Terms not specifically defined herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0031] Unless otherwise specified, terms used in the singular may also include the plural, and vice versa, where appropriate.

[0032] The term “about” when used in conjunction with a numeric value means a range of numeric values ​​that have a lower limit of 5%, 4%, 3%, 2%, or 1% smaller than the specified numeric value and an upper limit of 5%, 4%, 3%, 2%, or 1% larger than the specified numeric value.

[0033] When the term “and / or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0034] The terms “comprising,” “including,” and “containing” mean that the stated elements, values, or steps are included, but do not exclude any other elements, values, or steps. In this document, the terms “comprising,” “including,” and “containing” also cover situations where the text is substantially composed of or consists of the stated elements, values, or steps, unless the context clearly indicates that this is not applicable. For example, when referring to “comprising” an antibody variable region of a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0035] The term "consistent with..." means that it does not contain significant amounts of other components besides the defined components, for example, the amount of said other components is less than 10% w / w, preferably less than 5% w / w, more preferably less than 3% w / w, and most preferably less than 1% w / w.

[0036] The term "w / w" refers to the weight ratio.

[0037] The term "CD40L" refers to a ligand expressed on activated T cells. Other names for it are also known in the art, such as CD154, CD40 ligand (CD40L), CD40 anti-receptor (CD40CR), gp39, T-BAM, T-cell activation molecule, TRAF, TNF-associated activating protein (TRAP), and tumor necrosis factor ligand superfamily member 5 (TNFSF5). These terms are used interchangeably throughout the application. In some embodiments, the amino acid sequence of CD40L is shown in Uniport P29965. In some embodiments, CD40L comprises the sequence shown in SEQ ID NO: 17.

[0038] As used herein, the terms “anti-CD40L antibody,” “anti-CD40L,” “CD40L antibody,” or “antibody that binds to CD40L” refer to antibodies that are capable of binding with sufficient affinity to (human or cynomolgus monkey) CD40L or fragments thereof such that the antibody can be used as a diagnostic and / or therapeutic agent targeting (human or cynomolgus monkey) CD40L.

[0039] In this article, the term “antibody” is used in the broadest sense to refer to a protein that contains an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to complete antibodies and antigen-binding fragments of antibodies, full-length antibodies, multispecific antibodies, or bispecific antibodies.

[0040] The terms “full-length antibody,” “complete antibody,” and “intact antibody” are used interchangeably herein to refer to glycoproteins comprising at least two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions (complementarity-determining regions (CDRs) interspersed with more conserved regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions.

[0041] "Antibody fragment" refers to a molecule distinct from the intact antibody that contains a portion of the intact antibody and binds to the antigen bound by the intact antibody. The term "antigen-binding fragment" as used herein refers to a molecule distinct from the intact antibody that contains a portion of the intact antibody and binds to the antigen bound by the intact antibody.

[0042] The term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It comprises a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g., scFv); single-domain antibodies (sdAb) such as VHH, bivalent antibodies or fragments thereof, or camelid antibodies or diabody antibodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. Furthermore, digestion of a complete antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. The Fv fragment consists of the VL and VH domains of the antibody single arm. The two domains, VL and VH, of the Fv fragment can be encoded by independent genes, but they can also be generated as a single protein chain by using a recombinant approach and connecting the two domains with a synthetic linker peptide. In this single protein chain, the VL and VH regions pair to form a single-chain Fv (scFv).

[0043] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those 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 precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many known antibody CDR assignment schemes, including, for example: Chothia (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)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web at imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0044] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing methods or combinations thereof. A CDR may also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention). Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the numbering position 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)).

[0045] In some embodiments, the CDR of the heavy chain variable region of the antibody in this invention is determined according to Kabat or Chothia, or a combination of Kabat and Chothia. In some embodiments, the CDR of the light chain variable region of the antibody in this invention is determined according to Kabat.

[0046] In some embodiments, the CDR1 of the antibody heavy chain variable region in this invention is determined according to the Kabat and Chothia combination scheme (hereinafter referred to as "Kabat and Chothia combination", corresponding to H26-H32 in the Kabat numbering system), and the CDR2, CDR3 of the heavy chain variable region, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are determined according to the Kabat scheme.

[0047] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (such as a mouse monoclonal antibody) while exhibiting low immunogenicity when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portion of the antibody with its corresponding human portion (e.g., replacing the non-binding portions of the constant region and variable region with the corresponding portions of the human antibody).

[0048] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991, NIH Publication 91-3242. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Two Fc regions can dimerize to form a dimer Fc, and two different Fc regions can heterodimerize to form a heterodimeric Fc. In this document, the terms "Fc region," "Fc portion," and "dimeric Fc" do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include a hinge region or a partial hinge region at the N-terminus of the heavy chain constant region, such as the IgG1 hinge region or a partial IgG1 hinge region, for example, according to EU numbering, the sequence D221 to P230. In one embodiment, the human IgG heavy chain Fc region extends from Asp221 or from Cys226 or from Asp231 to the carboxyl terminus of the heavy chain.

[0049] In some implementations, amino acid substitutions are represented as (original amino acid, amino acid position, mutant amino acid). For example, when the substitution site is located in the C region, "L234A" means that leucine (L) at position 234 of EU is replaced by alanine (A). When referring to combinations of mutations, the mutations in the combination are connected by slashes. "L234A / L235A" indicates that both mutations L234A and L235A are included.

[0050] In some implementations, amino acid deletions are represented by △ + amino acid + mutation site, for example, △P329 represents the deletion of proline at position 329. Amino acid deletions can also be represented by "amino acid + mutation site + deletion", for example, the deletion of proline at position 329 can also be written as "P329 deletion".

[0051] The term "liquid formulation" refers to a formulation obtained by dissolving the defined components in an injectable solvent. Therefore, those skilled in the art will understand without question that the "liquid formulation" described herein necessarily includes injectable solvents. The term "injectable solvent" is preferably an aqueous injectable solvent, such as 0.9% sodium chloride injection, 5% glucose injection, and water for injection, with water for injection being more preferred.

[0052] The term "lyophilized formulation" refers to a solid composition obtained by freeze-drying a liquid formulation. The solid composition preferably has a water content of no more than about 3%, more preferably no more than about 1%. For example, the solid composition has a water content of about 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, or 3%.

[0053] The terms "reconstitution" or "redissolution" refer to the process of dissolving a lyophilized formulation in a suitable solvent (referred to as the "reconstitution solvent") to obtain a solution that can be applied to an individual. The reconstitution solvent is an injectable solvent suitable for direct application to an individual, preferably an aqueous injectable solvent, such as 0.9% sodium chloride injection, 5% glucose injection, or water for injection.

[0054] As used in this article, the term "stress conditions" refers to chemically and / or physically unfavorable environments that prevent protein drugs from maintaining stability, such as high temperature, high humidity, stirring or shaking, freeze-thaw cycles, etc. For example, "high temperature stress" refers to storing protein preparations at room temperature or even higher temperatures (e.g., 40°C ± 2°C) for a period of time. Stress tests can be used to examine the stability of antibody preparations and predict their shelf life.

[0055] The term "injection" refers to bolus or infusion, including but not limited to intravenous, intramuscular, intra-arterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, subcutaneous, intra-articular, and intraspinal injections, preferably intravenous injections, and particularly intravenous infusions. When used for intravenous infusion, if necessary, the formulations of the present invention can be reconstituted or diluted with a "reconstituted solvent," such as 0.9% sodium chloride injection and / or 5% glucose injection and / or water for injection.

[0056] The term "effective amount" refers to the amount of anti-CD40L antibody that produces the desired effect in an individual after administration of the lyophilized formulation of the present invention. The effective amount can be readily determined by a physician skilled in the art by taking into account a variety of factors, such as the individual's species, age, weight and general health condition, the specific disease involved, the severity of the disease, the individual's response, the specific antibody administered, the method of administration, and concomitant treatments.

[0057] The term "treatment" refers to stopping, alleviating, or reversing an existing disease or its symptoms, or slowing the progression of an existing disease or its symptoms.

[0058] The term "prevention" refers to administering medication to individuals at risk of developing a disease in order to prevent the occurrence or onset of the disease or its symptoms.

[0059] The term "individual" refers to an animal, particularly a mammal. The individual includes, but is not limited to, birds, cattle, sheep, cats, dogs, pigs, horses, rats, mice, guinea pigs, rabbits, monkeys, orangutans, and humans. In some embodiments, the individual is a human.

[0060] The term "pH adjuster" refers to an acidic or alkaline substance that can alter the pH value of a solution. In some embodiments, the pH adjuster is a pH buffer, i.e., a substance that, when a certain amount of acid or base is added, prevents a significant change in the pH of the solution, thereby maintaining the pH value within a desired range. For the purposes of this invention, the pH adjuster ensures that the solution formulations and lyophilized formulations of this invention have a pH value of 5.0-6.0 in the solution prior to lyophilization.

[0061] The term "protein protectant" refers to a pharmaceutically acceptable substance that helps maintain the stability of anti-CD40L antibody or its antigen-binding fragment during the preparation, transport, and storage of the liquid and lyophilized formulations of this invention.

[0062] The term "surfactant" refers to a substance that is amphiphilic due to the presence of both hydrophilic and lipophilic groups and can reduce surface tension. Commonly used surfactants are well known in the art, for example, as seen in *Complete Guide to Pharmaceutical Excipients*, edited by Luo Mingsheng and Gao Tianhui, 1st edition, 1995, Sichuan Science and Technology Press, and *Remington: The Science and Practice of Pharmacy*, 19th edition, 1995. In some embodiments, the surfactant is a nonionic surfactant.

[0063] The term "osmolarity regulator," also known as an isotonic agent, refers to a substance that brings the osmotic pressure of an injectable preparation close to that of animal (especially human) plasma and body fluids, thereby preventing hemolysis. Commonly used osmolarity regulators are well-known in the art; for example, they can be found in *Complete Compendium of Pharmaceutical Excipients*, edited by Luo Mingsheng and Gao Tianhui, 1st edition, 1995, Sichuan Science and Technology Press. For example, the osmolarity regulator used in the formulation of this invention can be selected from sucrose, mannitol, sorbitol, glycine, trehalose, arginine, glycerol, propylene glycol, sodium chloride, potassium iodide, sodium citrate, etc.

[0064] The term "pharmaceutically acceptable" means that there are no unacceptable toxicities, irritants, or side effects for animals, especially humans.

[0065] The term "parts by weight" is used to indicate the relative weight ratio between defined pharmaceutical components.

[0066] Implementation Plan Description

[0067] In a first aspect, the present invention provides a pharmaceutical formulation comprising an anti-CD40L antibody or an antigen-binding fragment thereof, a pH adjuster, a protein protectant, and a surfactant, wherein the formulation is a lyophilized formulation or a liquid formulation, and the liquid formulation has a pH value of 5.0-6.0.

[0068] The "anti-CD40L antibody or its antigen-binding fragment" described herein is the active substance of the pharmaceutical preparation of the present invention, and preferably the only active substance.

[0069] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), and a third heavy chain complementarity-determining region (HCDR3), as well as a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or respectively composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. The amino acid sequence composition shown in NO:9, for example, the CDR is determined by any CDR assignment scheme, such as Kabat, Chothia, IMGT, AbM or combinations thereof such as the Kabat and Chothia combination. For example, HCDR1 is determined according to the Kabat and Chothia combination scheme (hereinafter referred to as "Kabat and Chothia combination", corresponding to H26-H32 in the Kabat numbering system), and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are determined according to the Kabat scheme.

[0070] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19, or is composed of said sequence, or comprises the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19, or is composed of said sequence; or it comprises a light chain variable region (VL), wherein the light chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:10 or the nucleic acid sequence shown in SEQ ID NO:20, or is composed of said sequence, or comprises SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19, or is composed of said sequence; or comprises the amino acid sequence shown in SEQ ID NO:10 or the nucleic acid sequence shown in SEQ ID NO:20, or is composed of said sequence, or comprises the amino acid sequence shown in SEQ ID NO:10 or the nucleic acid sequence shown in SEQ ID NO:20, or is composed of said sequence, or comprises the amino acid sequence shown in SEQ ID NO:10 or the nucleic acid sequence shown in SEQ ID NO:20, or is composed of said sequence. The amino acid sequence shown in NO:10 or the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:20, or the sequence thereof.

[0071] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence encoded by the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence, or is composed of the amino acid sequence, and the light chain variable region comprises an amino acid sequence encoded by the amino acid sequence shown in SEQ ID NO:10 or the nucleic acid sequence shown in SEQ ID NO:20, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence, or is composed of the amino acid sequence.

[0072] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:10. In some embodiments, the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or is composed of the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:19, and the light chain variable region comprises or is composed of the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:20.

[0073] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment further comprises an Fc region, for example, wherein the Fc region is or is derived from human IgG Fc, for example, is or is derived from human IgG1 Fc, is or is derived from human IgG2 Fc, is or is derived from human IgG3 Fc or is derived from human IgG4 Fc; preferably, the Fc region comprises a mutation that reduces or eliminates effector function, and / or eliminates the binding affinity to FcγR while retaining the binding affinity to FcRn, preferably, the mutation is an L234A / L235A and / or P329 deletion mutation, for example, an L234A / L235A and P329 deletion mutation. In some embodiments, the Fc region

[0074] (i) Contains or consists of the amino acid sequence shown in SEQ ID NO:13 or 14;

[0075] (ii) Contains an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:13, for example, 95%, 96%, 97%, 98%, 99% or higher identity; or

[0076] (iii) Contains an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:14, such as 95%, 96%, 97%, 98%, 99% or higher, and contains L234A / L235A and P329 deletion mutations.

[0077] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment further comprises a heavy chain constant region, which is or is derived from a constant region of (human) IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the Fc and CH1 regions described herein. In some embodiments, the CH1 region comprises or is composed of the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0078] In some embodiments of the first aspect, the heavy chain constant region of the anti-CD40L antibody or its antigen-binding fragment...

[0079] (i) Contains or is composed of an amino acid sequence selected from or represented by SEQ ID NO:5 or 16;

[0080] (ii) Containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:16; or

[0081] (iii) Contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:5, and has L234A / L235A and P329 deletion mutations.

[0082] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment further comprises a light chain constant region, which is or is derived from the lambda or Kappa light chain constant region. Preferably, the light chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11; or comprises or is composed of the amino acid sequence shown in SEQ ID NO: 11.

[0083] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain, wherein the heavy chain

[0084] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:6; or

[0085] (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:6;

[0086] or

[0087] The anti-CD40L antibody or its antigen-binding fragment comprises a light chain, wherein the light chain

[0088] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12; or

[0089] (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:12.

[0090] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:6; and the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:12.

[0091] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:6, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0092] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment comprises two heavy chains and two light chains, wherein the heavy chains comprise or consist of the amino acid sequence shown in SEQ ID NO:6, and the light chains comprise or consist of the amino acid sequence shown in SEQ ID NO:12.

[0093] In some embodiments of the first aspect, the anti-CD40L antibody is a monoclonal antibody.

[0094] In some embodiments of the first aspect, the anti-CD40L antibody is a humanized antibody or a chimeric antibody.

[0095] In some embodiments of the first aspect, the anti-CD40L antibody is a full-length antibody.

[0096] In some embodiments of the first aspect, the antigen-binding fragment of the anti-CD40L antibody is selected from the following antibody fragments: Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibody, single-chain antibody (e.g., scFv); single-domain antibody (sdAb) such as VHH, bivalent antibody or fragment thereof, or camelid antibody or diabody antibody.

[0097] In some embodiments of the first aspect, the anti-CD40L antibody also encompasses multispecific antibodies, such as bispecific antibodies.

[0098] In some embodiments of the first aspect, the anti-CD40L antibody or its antigen-binding fragment is recombinantly expressed in host cells, such as mammalian cells like HEK 293 cells or CHO cells.

[0099] In some embodiments of the first aspect, the anti-CD40L antibody is the anti-CD40L antibody disclosed in PCT application number PCT / CN2024 / 090122, or an antigen-binding fragment thereof, which is incorporated herein by reference in its entirety as if its entire contents were set forth herein. Preferably, the anti-CD40L antibody is the anti-CD40L antibody Hz33B9C5 disclosed in PCT application number PCT / CN2024 / 090122, which consists of two heavy chain sequences shown in SEQ ID NO:6 and two light chain sequences shown in SEQ ID NO:12.

[0100] In a preferred embodiment of the first aspect, the anti-CD40L antibody refers to antibody A. Antibody A consists of 1326 amino acids, comprising two identical heavy chains and two identical light chains linked by 4 pairs of interchain disulfide bonds and 12 pairs of intrachain disulfide bonds. A structural schematic diagram of antibody A is shown in Figure 4. The heavy chain is γ1 type, consisting of 445 amino acids; the light chain is κ type, consisting of 218 amino acids. Antibody A contains two N-glycosylation sites, located at the 296th position of asparagine in both heavy chains, with the major glycoform being G0F. The molecular formula of antibody A is C0. 6414 H 9882 N 1702 O 2022 S 44 (Disulfide bonds are in a linked state), molecular weight is approximately 147.2 kDa (GOF / GOF sugar form), and theoretical extinction coefficient is 1.51 (mg / ml). -1 ·cm -1 .

[0101] The amino acid sequence of antibody A is shown in Figure 5, and its correctness has been confirmed by gene sequencing and amino acid sequence analysis. Antibody A underwent sequence modification in the Fc region to weaken its affinity for FcγRs and reduce ADCC and CDC effects. The modification sites, regions, and purposes are as follows:

[0102] In vitro affinity studies showed that antibody A had a strong affinity for human / cynomolgus monkey CD40L protein (human CD40L and cynomolgus monkey CD40L have the same extracellular segment sequence), at 4.68 pM. Antibody A did not bind to rat, mouse, or rabbit CD40L, nor to other human TNF family proteins such as TNF-alpha and OX40L, suggesting a low off-target risk. In vitro pharmacodynamic results showed that antibody A inhibited the binding of CD40L and CD40 proteins, blocking the activity of membrane-expressed CD40L binding to CD40. Both the inhibitory and blocking effects were comparable to those of the CD40L-targeting drug INX-021, indicating that antibody A has clear target selectivity and a significant inhibitory or blocking effect on CD40L-CD40 binding. The T cell-dependent activation pathway of B cells involves antigen endocytosis and presentation of CD40L by the major histocompatibility complex (MHCII). + After T cell recognition, B cells bind to CD40L ligands via the CD40 receptor on their surface, thereby differentiating into memory B cells and plasma cells. Antibody A can inhibit the proliferation of B cells and their ability to differentiate into plasma cells in vitro, suggesting that antibody A can inhibit B cell proliferation and differentiation by blocking the binding of CD40L to CD40.

[0103] The affinity of antibody A for FcRn is 10. -8 The affinity for FcγRI is on the order of M, and the affinity is in the range of 10. -7 The antibody A showed no significant binding to FcγRIIa, FcγRIIIa(V158), FcγRIIb, FcγRIIIb, FcγRIIIa(F158), or C1q, and no detectable ADCC (antibody-dependent cell-mediated cytotoxicity) or CDC (complement-dependent cytotoxicity) effects were observed. Antibody A dose-dependently inhibited EAE pathogenesis in a mouse model of experimental autoimmune encephalomyelitis (EAE) and dose-dependently inhibited anti-KLH antibody production in a KLH-induced in vivo T-cell-dependent antibody response mouse model.

[0104] Safety pharmacology studies using cynomolgus monkeys showed that when administered intravenously at doses up to 200 mg / kg, antibody A had no significant effect on blood pressure, electrocardiogram parameters, or respiratory rate in the monkeys, indicating that antibody A has excellent safety profile.

[0105] Based on preclinical studies of antibody A, it is anticipated that antibody A, along with other CD40L antibodies disclosed in international application PCT / CN2024 / 090122 and antibodies with similar structures, may be administered clinically at higher doses. For ease of administration, high-concentration (e.g., approximately 100 mg / ml) antibody formulations are required.

[0106] In the pharmaceutical formulation of the first aspect, the anti-CD40L antibody or its antigen-binding fragment may be present in a liquid formulation at a concentration of 50-100 mg / mL, for example 50, 60, 70, 80, 90 or 100 mg / mL, wherein the anti-CD40L antibody or its antigen-binding fragment constitutes 30-90% w / w, preferably 40-80% w / w, more preferably 50-60% w / w, for example about 58% w / w, of the lyophilized formulation.

[0107] In the pharmaceutical formulation of the first aspect, the formulation is a liquid formulation or a lyophilized formulation, and may contain 100 mg-1000 mg, for example 180 mg-550 mg, 200 mg-540 mg, 300 mg-600 mg, 300 mg-720 mg, 500 mg-1000 mg or 500 mg-800 mg, for example about 180 mg, 300 mg, 540 mg or 600 mg of the anti-CD40L antibody or its antigen-binding fragment.

[0108] In the pharmaceutical formulation of the first aspect, the amount of pH adjuster is such that the liquid formulation has a pH value of 5.0-6.0, preferably 5.0-5.5, and more preferably about 5.2. In some embodiments, the pH adjuster accounts for 0.05-1% w / w of the liquid formulation, for example 0.1-0.5% w / w, preferably about 0.2% w / w.

[0109] In the pharmaceutical formulation of the first aspect, a pH adjuster ensures that the solution of the lyophilized formulation prior to lyophilization has a pH of 5.0-6.0, preferably 5.0-5.5, and more preferably about 5.2. In some embodiments, the pH adjuster comprises 0.5-3% w / w of the lyophilized formulation, for example 1-2% w / w, preferably about 1.2% w / w. The pH of the reconstituted solution of the lyophilized formulation of the present invention depends on the solvent used for reconstitution. If the reconstituted solution is administered to an individual immediately, a pH between 4 and 9 is acceptable. If the reconstituted solution is stored for a period of time (e.g., stored at room temperature for 24 hours or stored at 0°C or lower (e.g., under freezing)) before administration to an individual, a pH between 5.0 and 6.0 is preferred.

[0110] In some embodiments of the first aspect, the pH adjuster may be any pharmaceutically acceptable pH-modifying substance suitable for use in injections. In some embodiments, the pH adjuster is selected from acetates (e.g., sodium acetate, potassium acetate), carbonates (sodium carbonate, sodium carbonate), bicarbonates (sodium bicarbonate), citrates (sodium citrate), amino acids (e.g., one or more amino acids selected from histidine, glycine, lysine, and arginine), amino acid salts (e.g., amino acid hydrochloride or sodium amino acid salts, such as histidine hydrochloride, arginine hydrochloride), phosphates (selected from one or more phosphates selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate), tris(hydroxymethyl)aminomethane (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), acetic acid, carbonic acid, citric acid, phosphoric acid, hydrochloric acid, and sodium hydroxide. In other embodiments, the pH adjuster is selected from sodium acetate, amino acids (e.g., one or more amino acids selected from histidine, glycine, lysine, and arginine), amino acid salts (e.g., amino acid hydrochloride or sodium amino acid salts, such as histidine hydrochloride and arginine hydrochloride), phosphates (selected from one or more phosphates selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate), tris(hydroxymethyl)aminomethane (Tris), and hydrochloric acid. In other embodiments, the pH adjuster is selected from sodium acetate, amino acids (e.g., one or more amino acids selected from histidine, glycine, lysine, arginine, and methionine), or amino acid salts (e.g., amino acid hydrochloride or sodium amino acid salts, such as histidine hydrochloride and arginine hydrochloride). In a preferred embodiment, the pH adjuster is selected from one or more of sodium acetate, hydrochloric acid, histidine, and histidine hydrochloride. In another preferred embodiment, the pH adjuster is histidine and a pharmaceutically acceptable salt of histidine (e.g., histidine hydrochloride), preferably in a weight ratio of 1:10 to 1:22, more preferably 1:12 to 1:20, more preferably 1:14 to 1:18, for example, about 1:16 of histidine and a pharmaceutically acceptable salt of histidine (e.g., histidine hydrochloride). More preferably, in the liquid formulation of the present invention, the pH adjuster is histidine and histidine hydrochloride in a weight ratio of about 1:16 (e.g., 1:16.3), and the total concentration of histidine in the liquid formulation is about 10 mM. In one embodiment, the formulation is a liquid formulation wherein the pH adjuster is 5-15 mM, for example 8-12 mM, for example about 10 mM of histidine. In one embodiment, the formulation is a liquid formulation wherein the pH adjuster is 10 mM histidine, the 10 mM histidine being composed of 0.12 mg / mL histidine and 1.94 mg / mL histidine hydrochloride.

[0111] Those skilled in the art will understand that the amino acids used herein are in their naturally occurring form. Preferably, the amino acids used herein are in the L-configuration.

[0112] In some embodiments of the first aspect, the protein protectant is selected from one or more of the following substances:

[0113] (1) Sugars, such as sucrose, lactose, trehalose, maltose, fructose, inulin oligosaccharides, glucan, glucose, maltodextrin, cyclodextrin, and dextran;

[0114] (2) Polyols, such as sorbitol, xylitol, glycerol, and ethylene glycol;

[0115] (3) Proteins, such as bovine serum albumin, human serum albumin, casein, and mucopolysaccharide proteins;

[0116] (4) Cellulose, such as carboxymethyl cellulose.

[0117] In some embodiments of the first aspect, the protein protectant is selected from one or more of the following substances:

[0118] (1) Sugars, such as sucrose, lactose, trehalose, maltose, fructose, inulin oligosaccharides, dextran, and maltodextrin;

[0119] (2) Polyols, such as sorbitol and xylitol.

[0120] In some embodiments of the first aspect, the protein protectant is selected from one or more of the following substances:

[0121] (1) Sugars, such as sucrose and trehalose;

[0122] (2) Polyols, such as sorbitol.

[0123] In some embodiments of the first aspect, the protein protectant is selected from one or more of sucrose, trehalose, and sorbitol.

[0124] In some embodiments of the first aspect, the protein protectant is sucrose.

[0125] In some embodiments of the first aspect, the protein protectant is present in the liquid formulation at a concentration of 15-90 mg / mL, for example 40-90 mg / mL, 50-80 mg / mL, preferably about 50 mg / mL. In a preferred embodiment, the protein protectant in the liquid formulation is sucrose, preferably present in the liquid formulation at a concentration of 50-80 mg / mL, preferably about 50 mg / mL.

[0126] In some embodiments of the first aspect, the protein protectant comprises 10-90% w / w, preferably 20-80% w / w, more preferably 30-50% w / w, for example about 40.61% w / w, of the lyophilized formulation. In a preferred embodiment, the protein protectant in the lyophilized formulation is sucrose, preferably comprising 30-50% w / w, for example about 40.61% w / w, of the lyophilized formulation.

[0127] In some embodiments of the first aspect, the surfactant may be an ionic surfactant or a nonionic surfactant. The ionic surfactant may be a cationic surfactant (e.g., dodecyltrimethylammonium chloride, octadecyltrimethylammonium chloride), anionic surfactant (e.g., sodium dodecylbenzenesulfonate, sodium dodecyl sulfonate), or amphoteric surfactant (e.g., methyl dodecylaminopropionate, sodium dodecylaminopropionate, sodium dodecylaminodiacetate, sodium octadecylaminopropionate, phosphatidylserine, lecithin, soybean lecithin). The nonionic surfactant may be selected from: sorbitan fatty acid esters, including polysorbates (e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85) and sorbitan esters (e.g., Span-20, Span-40, Span-60, Span-65, Span-80, Span-83, Span-85); polypropylene glycol ethylene oxide adducts, including poloxamers (e.g., poloxamer 188); and polyoxyethylene alkyl ethers, including Brij (e.g., Brij-30, Brij-35, Brij-52, Brij-56, Brij-58, Brij-76, Brij-28, Brij-92, Brij-96, Brij-98).

[0128] In some embodiments of the first aspect, the surfactant is a nonionic surfactant, which may be selected, for example, from sorbitan fatty acid esters, such as polysorbates (e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85) and sorbitan esters (e.g., Span-20, Span-40, Span-60, Span-65, Span-80, Span-83, Span-85).

[0129] In other embodiments of the first aspect, the surfactant is polysorbate.

[0130] In some embodiments of the first aspect, the surfactant is polysorbate-80 (e.g., Tween-80).

[0131] In some embodiments of the first aspect, the surfactant comprises 0.001-0.1% w / w, preferably 0.01-0.05% w / w, for example, about 0.03% w / w, of the liquid formulation; or the surfactant is present in the liquid formulation at a concentration of 0.05-1 mg / mL, preferably 0.1-0.5 mg / mL, for example, about 0.3 mg / mL. In some embodiments of the first aspect, the surfactant comprises 0.04-0.7% w / w, preferably 0.1-0.4% w / w, more preferably 0.1-0.3% w / w, for example, about 0.17% w / w, of the lyophilized formulation.

[0132] In addition to the components described above, the pharmaceutical formulation of the first aspect may optionally contain other components, such as lyophilization excipients and osmotic pressure regulators. The lyophilization excipients may be selected from glucose, dextran, mannitol, sorbitol, etc. The osmotic pressure regulators may be selected from mannitol, sorbitol, trehalose, sodium chloride, potassium iodide, glucose, etc. The amount of osmotic pressure regulator varies depending on the specific osmotic pressure regulator used, and is an amount that makes the osmotic pressure of the solution or reconstituted solution of the liquid formulation or lyophilized formulation of the present invention close to or equal to the normal osmotic pressure range of an individual. For humans, the normal plasma osmotic pressure range is 280-310 mOsm / (kg·H2O). Mannitol and glucose have the dual functions of lyophilization excipients and osmotic pressure regulators, and are therefore excellent excipients for protein lyophilized formulations. In one embodiment, the pharmaceutical formulation of the first aspect contains mannitol at a concentration of 10 mg / mL to 30 mg / mL, preferably 15 mg / mL to 25 mg / mL, more preferably about 20 mg / mL.

[0133] Those skilled in the art will understand that some substances can have multiple functions. For example, for the purposes of this invention, some amino acids can act as both pH adjusters and protein protectants; some sugar alcohols can act as both protein protectants and osmotic pressure regulators. However, the primary function of these substances with dual functions in this invention is the function specified in this invention.

[0134] In one embodiment of the first aspect, the pharmaceutical formulation of the present invention is a liquid formulation comprising:

[0135] (1) Anti-CD40L antibody or its antigen-binding fragment (e.g., anti-CD40L monoclonal antibody, preferably antibody A), wherein the anti-CD40L antibody or its antigen-binding fragment is preferably present in the liquid formulation at a concentration of 50-100 mg / mL, for example about 50, 60, 70, 80, 90 or 100 mg / mL;

[0136] (2) A protein protectant, wherein the protein protectant is preferably sucrose, and is preferably present in the liquid formulation at a concentration of 15-90 mg / mL, for example 40-90 mg / mL, 50-80 mg / mL, more preferably about 50 mg / mL;

[0137] (3) A surfactant, preferably a polysorbate (e.g., polysorbate 80), which is preferably present in the liquid formulation at a concentration of 0.05-1 mg / mL, more preferably 0.1-0.5 mg / mL, for example about 0.3 mg / mL;

[0138] (4) A pH adjuster, preferably selected from histidine and its pharmaceutically acceptable salts (e.g., histidine hydrochloride), wherein the amount of the pH adjuster is such that the liquid formulation has a pH of 5.0-6.0, preferably a pH of 5.0-5.5, and more preferably a pH of about 5.2; and

[0139] (5) Aqueous solvent for injection, selected from 0.9% sodium chloride injection, 5% glucose injection and water for injection, preferably water for injection;

[0140] Optionally, it also includes: (6) a pharmaceutically acceptable excipient, preferably mannitol, present in the liquid formulation at a concentration of 10 mg / mL to 30 mg / mL, preferably 15 mg / mL to 25 mg / mL, more preferably about 20 mg / mL.

[0141] In a preferred embodiment of the above-described liquid formulation, the pH adjuster is histidine and histidine hydrochloride. In a more preferred embodiment of the above-described liquid formulation, the pH adjuster is histidine and histidine hydrochloride in a weight ratio of 1:10 to 1:22, preferably 1:12 to 1:20, more preferably 1:14 to 1:18, for example, about 1:16. In a further preferred embodiment of the above-described liquid formulation, the pH adjuster is histidine and histidine hydrochloride, and the total histidine concentration produced in the liquid formulation is 5-15 mM, preferably about 10 mM.

[0142] In one embodiment of the first aspect, the pharmaceutical formulation of the present invention is a liquid formulation comprising about 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), about 10 mM histidine, about 50 mg / mL sucrose, about 20 mg / mL mannitol, about 0.3 mg / mL polysorbate 80, and about pH 5.2.

[0143] In another embodiment of the first aspect, the medicament of the present invention is a liquid formulation comprising about 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), about 10 mM histidine, about 80 mg / mL sucrose, about 0.3 mg / mL polysorbate 80, and a pH of about 5.2.

[0144] Preferably, in the pharmaceutical formulation of the present invention, the specified concentration of histidine is composed of histidine in free form and / or histidine in salt form (e.g., histidine hydrochloride). For example, 10 mM of histidine may be composed of 0.12 mg / mL histidine and 1.94 mg / mL histidine hydrochloride. Therefore, in a preferred embodiment, the pharmaceutical formulation of the present invention is a liquid formulation comprising about 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 mg / mL of histidine, about 1.94 mg / mL of histidine hydrochloride, about 50 mg / mL of sucrose, about 20 mg / mL of mannitol, about 0.3 mg / mL of polysorbate 80, and pH 5.2; in another preferred embodiment, the pharmaceutical formulation of the present invention is a liquid formulation comprising about 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 mg / mL of histidine, about 1.94 mg / mL of histidine hydrochloride, about 80 mg / mL of sucrose, about 0.3 mg / mL of polysorbate 80, and pH about 5.2.

[0145] In one embodiment of the present invention, the present invention provides a freeze-dried formulation prepared from the liquid formulation described in the first aspect by freeze-drying.

[0146] In one embodiment of the present invention, a lyophilized formulation is provided, comprising:

[0147] (1) 50-100 parts by weight, for example about 50, 60, 70, 80, 90 or 100 parts by weight of anti-CD40L antibody or its antigen-binding fragment (e.g. anti-CD40L monoclonal antibody, preferably antibody A);

[0148] (2) 15-90 parts by weight, for example 40-90 parts by weight, 50-80 parts by weight, more preferably about 50 parts by weight of a protein protectant (preferably sucrose);

[0149] (3) 0.05-1 parts by weight, more preferably 0.1-0.5 parts by weight, for example about 0.3 parts by weight of a surfactant (preferably polysorbate, for example polysorbate 80);

[0150] (4) 0.77-2.33 parts by weight, preferably 1.24-1.86 parts by weight, more preferably about 1.55 parts by weight, based on histidine, of a pH adjuster selected from histidine and pharmaceutically acceptable salts of histidine (e.g., histidine hydrochloride).

[0151] It may also optionally contain: (6) 10-30 parts by weight, preferably 15-25 parts by weight, more preferably about 20 parts by weight of a pharmaceutically acceptable excipient (preferably mannitol).

[0152] In a preferred embodiment of the above-described lyophilized formulation, the lyophilized formulation comprises (4) about 1.55 parts by weight of a pH adjuster based on histidine, said pH adjuster being histidine and histidine hydrochloride. In a more preferred embodiment of the above-described lyophilized formulation, said pH adjuster consists of about 0.12 parts by weight of histidine and about 1.94 parts by weight of histidine hydrochloride.

[0153] In one preferred embodiment, the pharmaceutical formulation of the present invention is a lyophilized formulation comprising about 100 parts by weight of an anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 parts by weight of histidine, about 1.94 parts by weight of histidine hydrochloride, about 50 parts by weight of sucrose, about 20 parts by weight of mannitol, and about 0.3 parts by weight of polysorbate 80; in another preferred embodiment, the pharmaceutical formulation of the present invention is a lyophilized formulation comprising about 100 parts by weight of an anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 parts by weight of histidine, about 1.94 parts by weight of histidine hydrochloride, about 80 parts by weight of sucrose, and about 0.3 parts by weight of polysorbate 80.

[0154] In the pharmaceutical formulation of the present invention, the histidine is preferably L-histidine.

[0155] The pharmaceutical formulations of the present invention, particularly the lyophilized formulations, preferably contain about 600 mg of the anti-CD40L antibody or its antigen-binding fragment (e.g., antibody A) per vial.

[0156] Those skilled in the art will also understand that the substances contained in the lyophilized formulations of the present invention are all pharmaceutically acceptable.

[0157] The liquid formulation of the present invention must be stored at low temperatures, such as at ≤0°C, or at ≤20°C, or at ≤40°C.

[0158] The lyophilized formulation of the present invention is reconstituted using a reconstituted solvent before individual administration. The reconstituted solvent may be 0.9% sodium chloride injection, 5% glucose injection, water for injection, etc. The pH of the reconstituted solution varies depending on the reconstituted solvent. If administered to an individual immediately after reconstitution, a pH value between 4 and 9 is acceptable. If the reconstituted solution is stored for a period of time (e.g., stored at room temperature for 24 hours or at 0°C or lower (e.g., under freezing)) before administration to an individual, it is preferable to maintain the pH value of the reconstituted solution between 5.0 and 6.0.

[0159] The lyophilized formulation of the present invention, after reconstruction, forms a solution for injection, particularly by intravenous injection, preferably by intravenous infusion.

[0160] The liquid formulation of the present invention can be stably stored under frozen conditions, for example, for at least 12 months or longer, such as 36 months.

[0161] The lyophilized formulations of the present invention can be stored stably for a long period of time at a temperature ≤40°C, for example, for at least 12 months or longer, such as 36 months. In one embodiment, the lyophilized formulations of the present invention can be stored stably for 36 months under refrigeration conditions (i.e., 2°C to 8°C) or stably for 12 months at room temperature.

[0162] The liquid and lyophilized formulations of the present invention can be used to prevent or treat diseases or conditions related to inappropriate activation of the CD40L / CD40-mediated pathway.

[0163] In a second aspect, the present invention provides a pillbox containing the pharmaceutical preparation described in the first aspect, and instructions for using the preparation to prevent or treat an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway.

[0164] In some embodiments of the second aspect, the present invention provides a kit containing the lyophilized formulation described in the first aspect and instructions for using the formulation to prevent or treat an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway. The kit also contains a reconstituted solvent in a separate container, preferably selected from 0.9% sodium chloride injection, 5% glucose injection, and water for injection.

[0165] In a third aspect, the present invention provides a pre-filled syringe containing the pharmaceutical preparation described in the first aspect.

[0166] In a fourth aspect, the present invention provides a method for preparing the pharmaceutical formulation described in the first aspect, the method comprising the following steps:

[0167] (1) Dissolve the pH adjuster and protein protectant in an injectable solvent, add the anti-CD40L antibody or its antigen-binding fragment, and optionally adjust the pH value to pH 5.0-6.0;

[0168] (2) Add a surfactant to obtain the liquid formulation;

[0169] Optionally, (3) the liquid formulation obtained in step (2) is freeze-dried to obtain the freeze-dried formulation.

[0170] In the method of the fourth aspect, the order of addition of each component may be changed as appropriate. These changes are obvious to those skilled in the art and are all within the scope of protection of this application.

[0171] In some embodiments of the fourth aspect, the freeze-drying described in step (3) includes the following steps: (a) pre-freezing, for example, first pre-freezing at atmospheric pressure at -3°C to 7°C, preferably 0°C to 4°C, more preferably about 2°C for about 1 hour, and then pre-freezing at atmospheric pressure at -50°C to -40°C, preferably -47°C to -43°C, more preferably about -45°C for about 4 hours; (b) vacuuming, for example, vacuuming to about 32 Pa; (c) performing a primary drying at vacuum at -25°C to -15°C, preferably -22°C to -18°C, more preferably about -20°C for about 36 hours; and (d) performing a secondary drying at vacuum at 20°C to 30°C, preferably 23°C to 27°C, more preferably about 25°C for about 18 hours.

[0172] In some embodiments of the fourth aspect, the method further includes step (4): purging the freeze-dried product of step (3) with nitrogen to 750-850 mbar and then plugging it.

[0173] In a fifth aspect, the present invention provides a pharmaceutical preparation as described in the first aspect for the prevention or treatment of diseases or conditions associated with inappropriate activation of the CD40L / CD40-mediated pathway.

[0174] In a sixth aspect, the present invention provides the use of the pharmaceutical preparations described in the first aspect for the prevention or treatment of diseases or conditions related to inappropriate activation of the CD40L / CD40-mediated pathway.

[0175] In a seventh aspect, the present invention provides a method for preventing or treating an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway, the method comprising injecting the individual with an effective amount of the pharmaceutical preparation described in the first aspect.

[0176] In all the foregoing aspects, the diseases or conditions associated with inappropriate activation of the CD40L / CD40-mediated pathway may be selected from: autoimmune diseases such as primary Sjögren's syndrome. Syndrome), systemic lupus erythematosus, multiple sclerosis, lupus nephritis, immune thrombocytopenic purpura (ITP), Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, psoriasis, myasthenia gravis, graft-versus-host disease, graft rejection and inflammatory bowel disease (IBD), neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), progressive bulbar palsy and spinal muscular atrophy, tumors, atherosclerosis, coronary heart disease, type 1 diabetes, asthma, allergic reactions and immune responses to recombinant drug products such as hemophilia factor VII.

[0177] In a preferred embodiment, the disease or condition associated with inappropriate activation of the CD40L / CD40-mediated pathway is selected from autoimmune diseases such as primary Sjögren's syndrome, systemic lupus erythematosus, multiple sclerosis, lupus nephritis, immune thrombocytopenic purpura (ITP), Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, psoriasis, myasthenia gravis, graft-versus-host disease, graft rejection, and inflammatory bowel disease (IBD).

[0178] In a more preferred embodiment, the disease or condition associated with inappropriate activation of the CD40L / CD40-mediated pathway is selected from autoimmune diseases, such as primary Sjögren's syndrome and systemic lupus erythematosus. Detailed Implementation Plan

[0179] Implementation Scheme 1. A pharmaceutical formulation comprising an anti-CD40L antibody or an antigen-binding fragment thereof, a pH adjuster, a protein protectant, and a surfactant, wherein the formulation is a lyophilized formulation or a liquid formulation, and the liquid formulation has a pH value of 5.0-6.0, preferably a pH value of 5.0-5.5, more preferably a pH value of about 5.2, wherein the anti-CD40L antibody or the antigen-binding fragment thereof comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), and a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or respectively composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. The amino acid sequences shown in NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 are composed of amino acid sequences.

[0180] Implementation Scheme 2. The pharmaceutical formulation according to Implementation Scheme 1, wherein the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence encoded by the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19, or comprises an amino acid sequence encoded by the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19, or comprises an amino acid sequence encoded by the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19; or comprises a light chain variable region (VL), wherein the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:10 or the nucleic acid sequence shown in SEQ ID NO:20, or comprises an amino acid sequence encoded by ... The amino acid sequence encoded by the amino acid sequence shown in NO:10 or the nucleic acid sequence shown in SEQ ID NO:20, or the amino acid sequence composed of said sequences.

[0181] Implementation Scheme 3. The pharmaceutical formulation according to any one of Implementation Schemes 1-2, wherein the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence encoded by the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19 or is composed of the amino acid sequence shown in SEQ ID NO:20 or is composed of the amino acid sequence shown in SEQ ID NO:10 or the nucleic acid sequence shown in SEQ ID NO:20.

[0182] Implementation Scheme 4. The pharmaceutical formulation according to any one of Implementation Schemes 1-3, wherein the anti-CD40L antibody or its antigen-binding fragment further comprises an Fc region, for example, wherein the Fc region is or is derived from human IgG Fc, for example, is or is derived from human IgG1 Fc, is or is derived from human IgG2 Fc, is or is derived from human IgG3 Fc or is derived from human IgG4 Fc; preferably, the Fc region comprises a mutation that reduces or eliminates effector function, and / or eliminates the binding affinity to FcγR while retaining the binding affinity to FcRn, preferably, the mutation is an L234A / L235A and / or P329 deletion mutation, preferably, the Fc region

[0183] (i) Contains or consists of the amino acid sequence shown in SEQ ID NO:13 or 14;

[0184] (ii) Contains an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:13, for example, 95%, 96%, 97%, 99% or higher identity; or

[0185] (iii) Contains an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:14, such as 95%, 96%, 97%, 99% or higher, and contains L234A / L235A and P329 deletion mutations.

[0186] Implementation Scheme 5. The pharmaceutical formulation according to any one of Implementation Schemes 1-4, wherein the anti-CD40L antibody or its antigen-binding fragment further comprises a heavy chain constant region, said heavy chain constant region being or derived from a constant region of (human) IgG1, IgG2, IgG3 or IgG4, preferably, said heavy chain constant region

[0187] (i) Contains or is composed of an amino acid sequence selected from or represented by SEQ ID NO:5 or 16;

[0188] (ii) comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:16; or

[0189] (iii) Contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:5, and has L234A / L235A and P329 deletion mutations;

[0190] and / or

[0191] The anti-CD40L antibody or its antigen-binding fragment further comprises a light chain constant region, which is or is derived from the light chain constant region of (human) lambda or Kappa. Preferably, the light chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:11; or comprises or is composed of the amino acid sequence shown in SEQ ID NO:11.

[0192] Implementation Scheme 6. The pharmaceutical formulation according to any one of Implementation Schemes 1-5, wherein the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain, wherein the heavy chain

[0193] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:6; or

[0194] (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:6;

[0195] or

[0196] The anti-CD40L antibody or its antigen-binding fragment comprises a light chain, wherein the light chain

[0197] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12; or

[0198] (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:12.

[0199] Implementation Scheme 7. The pharmaceutical formulation according to any one of Implementation Schemes 1-6, wherein the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:6, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0200] Implementation Scheme 8. The pharmaceutical preparation according to any one of Implementation Schemes 1-7, wherein the anti-CD40L antibody is a humanized antibody or a chimeric antibody.

[0201] Implementation Scheme 9. The pharmaceutical formulation according to any one of Implementation Schemes 1-8, wherein the antigen-binding fragment of the anti-CD40L antibody is selected from the following antibody fragments: Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibody, single-chain antibody (e.g., scFv); single-domain antibody (sdAb) such as VHH, bivalent antibody or fragment thereof, or camelid antibody or diabody antibody.

[0202] Implementation Scheme 10. The pharmaceutical preparation according to any one of Implementation Schemes 1-9, wherein the anti-CD40L antibody is composed of two heavy chains and two light chains, wherein the heavy chains are composed of the amino acid sequence shown in SEQ ID NO:6 and the light chains are composed of the amino acid sequence shown in SEQ ID NO:12, preferably the amino acid sequence of the anti-CD40L antibody is shown in Figure 5.

[0203] Implementation Scheme 11. A pharmaceutical formulation according to any one of Implementation Schemes 1-10, wherein the formulation is a liquid formulation and the anti-CD40L antibody or its antigen-binding fragment is present in the liquid formulation at a concentration of 50-100 mg / mL, for example about 50, 60, 70, 80, 90 or 100 mg / mL, or wherein the formulation is a lyophilized formulation and the anti-CD40L antibody or its antigen-binding fragment constitutes 30-90% w / w, preferably 40-80% w / w, more preferably 50-60% w / w, for example about 58% w / w, of the lyophilized formulation.

[0204] Implementation Scheme 12. A pharmaceutical formulation according to any one of Implementation Schemes 1-11, wherein the formulation comprises 100 mg-1000 mg, for example 180 mg-550 mg, 200 mg-540 mg, 300 mg-600 mg, 300 mg-720 mg, 500 mg-1000 mg or 500 mg-800 mg, for example about 180 mg, 300 mg, 540 mg or 600 mg of the anti-CD40L antibody or its antigen-binding fragment.

[0205] Implementation Scheme 13. The pharmaceutical preparation according to any one of Implementation Schemes 1-12, wherein

[0206] The pH adjuster is selected from one or more of the following: acetate (e.g., sodium acetate, potassium acetate), carbonate (e.g., sodium carbonate, sodium carbonate), bicarbonate (e.g., sodium bicarbonate), citrate (e.g., sodium citrate), amino acids (e.g., histidine, glycine, lysine, and arginine), amino acid salts (e.g., amino acid hydrochloride or sodium amino acid salts, such as histidine hydrochloride, arginine hydrochloride, monosodium glutamate), phosphates (e.g., sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate), tris(hydroxymethyl)aminomethane (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), acetic acid, carbonic acid, citric acid, phosphoric acid, hydrochloric acid, and sodium hydroxide.

[0207] or

[0208] The pH adjuster is selected from one or more of sodium acetate, amino acids (e.g., histidine, glycine, lysine, and arginine), amino acid salts (e.g., amino acid hydrochloride or sodium amino acid salts, such as histidine hydrochloride, arginine hydrochloride), phosphates (e.g., sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate), tris(hydroxymethyl)aminomethane (Tris), and hydrochloric acid.

[0209] or

[0210] The pH adjuster is selected from one or more of sodium acetate, amino acids (e.g., histidine, glycine, lysine, arginine, and methionine), amino acid hydrochlorides (e.g., histidine hydrochloride, arginine hydrochloride), and hydrochloric acid.

[0211] or

[0212] The pH adjuster is selected from one or more of sodium acetate, hydrochloric acid, histidine, and histidine hydrochloride.

[0213] or

[0214] The pH adjuster is histidine and histidine hydrochloride. Preferably, the pH adjuster is histidine and histidine hydrochloride in a weight ratio of 1:10 to 1:22, more preferably 1:12 to 1:20, more preferably 1:14 to 1:18, for example about 1:16.

[0215] Implementation Scheme 14. The pharmaceutical preparation according to any one of Implementation Schemes 1-13, wherein,

[0216] The pharmaceutical preparation is a liquid preparation, the pH adjuster is histidine and histidine hydrochloride in a weight ratio of about 1:16, and the total concentration of histidine in the liquid preparation is about 10 mM, preferably the total concentration of histidine of about 10 mM consists of about 0.12 mg / mL free histidine and about 1.94 mg / mL histidine hydrochloride;

[0217] or

[0218] The pharmaceutical formulation is a lyophilized formulation, and the pH adjuster is histidine and histidine hydrochloride in a weight ratio of 1:10 to 1:22, preferably 1:12 to 1:20, more preferably 1:14 to 1:18, for example about 1:16, and the pH adjuster accounts for 0.5-3% w / w, for example 1-2% w / w, preferably about 1.2% w / w, of the lyophilized formulation.

[0219] Implementation Scheme 15. The pharmaceutical preparation according to any one of Implementation Schemes 1-14, wherein

[0220] The protein protectant is selected from one or more of the following substances:

[0221] (1) Sugars, such as sucrose, lactose, trehalose, maltose, fructose, inulin oligosaccharides, glucan, glucose, maltodextrin, cyclodextrin, and dextran;

[0222] (2) Polyols, such as sorbitol, xylitol, glycerol, and ethylene glycol;

[0223] (3) Proteins, such as bovine serum albumin, human serum albumin, casein, and mucopolysaccharide proteins;

[0224] (4) Cellulose, such as carboxymethyl cellulose,

[0225] or

[0226] The protein protectant is selected from one or more of the following substances:

[0227] (1) Sugars, such as sucrose, lactose, trehalose, maltose, fructose, inulin oligosaccharides, dextran, and maltodextrin;

[0228] (2) Polyols, such as sorbitol and xylitol,

[0229] or

[0230] The protein protectant is selected from one or more of the following substances:

[0231] (1) Sugars, such as sucrose and trehalose;

[0232] (2) Polyols, such as sorbitol,

[0233] or

[0234] The protein protectant is selected from one or more of sucrose, trehalose, and sorbitol;

[0235] or

[0236] The protein protectant is sucrose.

[0237] Implementation Scheme 16. The pharmaceutical preparation according to any one of Implementation Schemes 1-15, wherein,

[0238] The formulation is a liquid formulation, wherein the protein protectant comprises 1-15% w / w, preferably 2-10% w / w, more preferably 6-8% w / w, for example about 6.67% w / w; or the formulation is a liquid formulation, wherein the protein protectant is present in the liquid formulation at a concentration of 15-90 mg / mL, preferably 40-90 mg / mL, 50-80 mg / mL, more preferably about 50 mg / mL;

[0239] or

[0240] The formulation is a lyophilized formulation, wherein the protein protectant accounts for 10-90% w / w of the lyophilized formulation, preferably 20-80% w / w, more preferably 30-50% w / w, for example about 40.61% w / w.

[0241] Implementation Scheme 17. The pharmaceutical preparation according to any one of Implementation Schemes 1-16, wherein,

[0242] The surfactant may be a nonionic surfactant or an ionic surfactant. The nonionic surfactant may be selected from: sorbitan fatty acid esters, including polysorbates (e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85) and sorbitan esters (e.g., Span-20, Span-40, Span-60, Span-65, Span-80, Span-83, Span-85); polypropylene glycol ethylene oxide adducts, including poloxamer derivatives (e.g., poloxamer 188); and polyoxyethylene alkyl ethers, including benzyl ethers (Brij derivatives) (e.g., Brij-3). 0, Brij-35, Brij-52, Brij-56, Brij-58, Brij-76, Brij-28, Brij-92, Brij-96, Brij-98), wherein the ionic surfactant can be a cationic surfactant (e.g., dodecyltrimethylammonium chloride, octadecyltrimethylammonium chloride), anionic surfactant (e.g., sodium dodecylbenzenesulfonate, sodium dodecyl sulfonate), or amphoteric surfactant (e.g., methyl dodecylaminopropionate, sodium dodecylaminopropionate, sodium dodecylaminodiacetate, sodium octadecylaminopropionate, phosphatidylserine, lecithin, soybean lecithin),

[0243] or

[0244] The surfactant is a nonionic surfactant selected from: sorbitan fatty acid esters, including polysorbates (e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85) and sorbitan esters (e.g., Span-20, Span-40, Span-60, Span-65, Span-80, Span-83, Span-85); polypropylene glycol ethylene oxide adducts, including poloxamer derivatives (e.g., poloxamer 188); and polyoxyethylene alkyl ethers, including benzyl ethers (Brij) (e.g., Brij-30, Brij-35, Brij-52, Brij-56, Brij-58, Brij-76, Brij-28, Brij-92, Brij-96, Brij-98).

[0245] or

[0246] The surfactant is a nonionic surfactant selected from sorbitan fatty acid esters, such as polysorbates (e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85) and sorbitan esters (e.g., Span-20, Span-40, Span-60, Span-65, Span-80, Span-83, Span-85).

[0247] or

[0248] The surfactant is polysorbate, preferably polysorbate-80.

[0249] Implementation Scheme 18. The pharmaceutical preparation according to any one of Implementation Schemes 1-17, wherein,

[0250] The formulation is a liquid formulation, wherein the surfactant accounts for 0.001-0.1% w / w, preferably 0.01-0.05% w / w, for example about 0.03% w / w; or the formulation is a liquid formulation, wherein the surfactant is present in the liquid formulation at a concentration of 0.1 mg / mL to 0.5 mg / mL, preferably 0.2 mg / mL to 0.4 mg / mL, more preferably about 0.3 mg / mL.

[0251] or

[0252] The formulation is a lyophilized formulation, and the surfactant accounts for 0.04-0.7% w / w of the lyophilized formulation, preferably 0.1-0.4% w / w, more preferably 0.1-0.3% w / w, for example about 0.17% w / w.

[0253] Implementation Scheme 19. The pharmaceutical preparation according to Implementation Scheme 1, wherein,

[0254] The formulation is a liquid formulation comprising (1) an anti-CD40L antibody or its antigen-binding fragment (e.g., an anti-CD40L monoclonal antibody, preferably antibody A), wherein the anti-CD40L antibody or its antigen-binding fragment is preferably present in the liquid formulation at a concentration of 50-100 mg / mL, for example, about 50, 60, 70, 80, 90 or 100 mg / mL; (2) a protein protectant, wherein the protein protectant is preferably sucrose, and is preferably present in the liquid formulation at a concentration of 15-90 mg / mL, for example, 40-90 mg / mL, 50-80 mg / mL, more preferably about 50 mg / mL; and (3) a surfactant, wherein the surfactant is preferably polysorbate (e.g., polysorbate 80), and is preferably present in the liquid formulation at a concentration of 0.05-1 mg / mL. More preferably, it is present at a concentration of 0.1-0.5 mg / mL, for example, about 0.3 mg / mL; (4) a pH adjuster, preferably selected from histidine and its pharmaceutically acceptable salts (e.g., histidine hydrochloride), the amount of which causes the liquid formulation to have a pH of 5.0-6.0, preferably 5.0-5.5, more preferably about 5.2; (5) an aqueous solvent for injection, selected from 0.9% sodium chloride injection, 5% glucose injection and water for injection, preferably water for injection; and optionally also includes (6) a pharmaceutically acceptable excipient, preferably mannitol, present in the liquid formulation at a concentration of 10 mg / mL to 30 mg / mL, preferably 15 mg / mL to 25 mg / mL, more preferably about 20 mg / mL;

[0255] or

[0256] The formulation is a lyophilized formulation comprising (1) 50-100 parts by weight, for example about 50, 60, 70, 80, 90 or 100 parts by weight of an anti-CD40L antibody or its antigen-binding fragment (e.g., an anti-CD40L monoclonal antibody, preferably antibody A); (2) 15-90 parts by weight, for example 40-90 parts by weight, 50-80 parts by weight, more preferably about 50 parts by weight of a protein protectant (preferably sucrose); and (3) 0.05-1 parts by weight, more preferably 0.1-0.5 parts by weight, for example about 0. 3 parts by weight of a surfactant (preferably polysorbate, such as polysorbate 80); (4) 0.77-2.33 parts by weight, preferably 1.24-1.86 parts by weight, more preferably about 1.55 parts by weight, based on histidine, said pH adjuster being selected from histidine and pharmaceutically acceptable salts of histidine (e.g., histidine hydrochloride); and optionally also comprising (6) 10-30 parts by weight, preferably 15-25 parts by weight, more preferably about 20 parts by weight of a pharmaceutically acceptable excipient (preferably mannitol).

[0257] Implementation Scheme 20. The pharmaceutical preparation according to Implementation Scheme 1, wherein

[0258] The formulation is a liquid formulation containing approximately 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), approximately 10 mM histidine, approximately 50 mg / mL sucrose, approximately 20 mg / mL mannitol, approximately 0.3 mg / mL polysorbate 80, and a pH of approximately 5.2.

[0259] or

[0260] The formulation is a liquid formulation containing approximately 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), approximately 10 mM histidine, approximately 80 mg / mL sucrose, approximately 0.3 mg / mL polysorbate 80, and a pH of approximately 5.2.

[0261] or

[0262] The formulation is a lyophilized formulation containing about 100 parts by weight of anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 parts by weight of histidine, about 1.94 parts by weight of histidine hydrochloride, about 50 parts by weight of sucrose, about 20 parts by weight of mannitol, and about 0.3 parts by weight of polysorbate 80.

[0263] or

[0264] The formulation is a lyophilized formulation containing about 100 parts by weight of anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 parts by weight of histidine, about 1.94 parts by weight of histidine hydrochloride, about 80 parts by weight of sucrose, and about 0.3 parts by weight of polysorbate 80.

[0265] or

[0266] The formulation is a liquid formulation containing approximately 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), approximately 10 mM histidine, approximately 20 mg / mL sucrose, approximately 40 mg / mL mannitol, approximately 0.3 mg / mL polysorbate 80, and a pH of approximately 5.2.

[0267] or

[0268] The formulation is a lyophilized formulation containing about 100 parts by weight of anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 parts by weight of histidine, about 1.94 parts by weight of histidine hydrochloride, about 20 parts by weight of sucrose, about 40 parts by weight of mannitol, and about 0.3 parts by weight of polysorbate 80.

[0269] Implementation Scheme 21. The pharmaceutical formulation according to Implementation Scheme 20, wherein the formulation is a liquid formulation, and the approximately 10 mM histidine consists of approximately 0.12 mg / mL histidine and approximately 1.94 mg / mL histidine hydrochloride.

[0270] Implementation Scheme 22. The pharmaceutical preparation according to any one of Implementation Schemes 1-21, wherein the preparation contains about 600 mg of antibody.

[0271] Implementation Scheme 23. A pillbox comprising a pharmaceutical preparation according to any one of Implementation Schemes 1-22 and instructions for using the preparation to prevent or treat an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway.

[0272] Implementation Scheme 24. A kit according to Implementation Scheme 23, wherein the kit contains a pharmaceutical preparation according to any one of Implementation Schemes 1-22 and instructions for using the preparation to prevent or treat an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway, the preparation being a lyophilized preparation, and the kit further containing a reconstitution solvent in a separate container, preferably selected from 0.9% sodium chloride injection, 5% glucose injection, and water for injection.

[0273] Implementation Scheme 25. A pre-filled syringe containing a pharmaceutical preparation as described in any one of Implementation Schemes 1-22.

[0274] Implementation Scheme 26. A method for preparing a pharmaceutical preparation according to any one of Implementation Schemes 1-22, the method comprising the following steps:

[0275] (1) Dissolve the pH adjuster and protein protectant in an injectable solvent, add the anti-CD40L antibody or its antigen-binding fragment, and optionally adjust the pH value to pH 5.0-6.0;

[0276] (2) Add a surfactant to obtain the liquid formulation;

[0277] Optionally, (3) the liquid formulation obtained in step (2) is freeze-dried to obtain the freeze-dried formulation.

[0278] Implementation Scheme 27. The method according to Implementation Scheme 26, wherein the freeze drying in step (3) comprises the following steps: (a) pre-freezing, for example, first pre-freezing at atmospheric pressure at -3°C to 7°C, preferably at 0°C to 4°C, more preferably at about 2°C for about 1 hour, and then pre-freezing at atmospheric pressure at -50°C to -40°C, preferably at -47°C to -43°C, more preferably at about -45°C for about 4 hours; (b) vacuuming, for example, vacuuming to about 32 Pa; (c) performing a first drying at vacuum at -25°C to -15°C, preferably at -22°C to -18°C, more preferably at about -20°C for about 36 hours; (d) performing a second drying at vacuum at 20°C to 30°C, preferably at 23°C to 27°C, more preferably at about 25°C for about 18 hours.

[0279] Implementation Scheme 28. The method according to Implementation Scheme 26 or 27, wherein the method includes steps (1) to (3) and further includes step (4):

[0280] (4) Purge the freeze-dried product from step (3) with nitrogen to 750-850 mbar and then plug it.

[0281] Implementation Scheme 29. A pharmaceutical preparation according to any one of Implementation Schemes 1-22, for the prevention or treatment of diseases or conditions related to inappropriate activation of the CD40L / CD40-mediated pathway.

[0282] Implementation Scheme 30. A method for preventing or treating an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway, the method comprising injecting the individual with an effective amount of the pharmaceutical preparation described in any one of Implementation Schemes 1-22.

[0283] Implementation Scheme 31. The pharmaceutical formulation according to Implementation Scheme 29 or the method according to Implementation Scheme 30, wherein the disease or condition associated with inappropriate activation of the CD40L / CD40-mediated pathway is selected from: autoimmune diseases such as primary Sjögren's syndrome. Syndrome), systemic lupus erythematosus, multiple sclerosis, lupus nephritis, immune thrombocytopenic purpura (ITP), Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, psoriasis, myasthenia gravis, graft-versus-host disease, graft rejection and inflammatory bowel disease (IBD), neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), progressive bulbar palsy and spinal muscular atrophy, tumors, atherosclerosis, coronary heart disease, type 1 diabetes, asthma, allergic reactions and immune responses to recombinant drug products such as hemophilia factor VII.

[0284] Implementation Scheme 32. The pharmaceutical preparation according to Implementation Scheme 29 or the method according to Implementation Scheme 30, wherein the disease or condition associated with inappropriate activation of the CD40L / CD40-mediated pathway is selected from autoimmune diseases, such as primary Sjögren's syndrome, systemic lupus erythematosus, multiple sclerosis, lupus nephritis, immune thrombocytopenic purpura (ITP), Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, psoriasis, myasthenia gravis, graft-versus-host disease, graft rejection, and inflammatory bowel disease (IBD).

[0285] Implementation Scheme 33. The pharmaceutical formulation according to Implementation Scheme 29 or the method according to Implementation Scheme 30, wherein the disease or condition associated with inappropriate activation of the CD40L / CD40-mediated pathway is selected from autoimmune diseases, such as primary Sjögren's syndrome and systemic lupus erythematosus.

[0286] Example

[0287] The following examples are provided to illustrate the invention and should not be construed as limiting the scope of the appended claims in any way.

[0288] Example 1: Pre-prescription study

[0289] 1. Experimental Design

[0290] This study primarily investigated the effect of pH on the stability of antibody A. Buffer solutions containing 1.55 mg / mL histidine and 50.0 mg / mL sorbitol were prepared, and the pH was adjusted to 5.0, 5.5, 6.0, 6.5, and 7.0 using dilute hydrochloric acid. Antibody A was then ultrafiltered into buffer solutions at different pH values ​​to adjust the antibody A concentration to approximately 100 mg / mL. Polysorbate 80 was added to a final concentration of 0.3 mg / mL. The samples were filtered, dispensed into vials, and capped. The stability of the obtained samples was investigated at 40℃ ± 2℃, as detailed in Table 1.

[0291] Table 1. Experimental Scheme Note: (1) √ indicates that the sample was taken at this point. (2) After sampling at the specified time point, all samples should be frozen at -40℃ and thawed as needed before being sent for testing.

[0292] 2. Experimental Results

[0293] The experimental results are shown in Table 2, and the trend of change is shown in Figure 1.

[0294] Table 2. Results of Pre-Prescription Studies Note: (a) The standard for acceptable appearance is "clear to slightly opalescent, colorless to slightly yellow liquid, free of foreign matter"; (b) The standard for acceptable visible foreign matter is "free of foreign matter"; (c) *Activity is controlled by T0; (d) N / A indicates not tested; (e) The pH 7.0 sample was not tested due to its unacceptable appearance.

[0295] The results showed that after being stored at 40℃±2℃ for 4 weeks, samples at pH 6.5 and pH 7.0 exhibited turbidity, while samples at pH 5.0 to pH 6.0 showed acceptable appearance and no visible foreign matter. The protein content, pH value, and relative activity of samples at pH 5.0 to pH 6.0 did not change significantly. At all pH values, the purity changes determined by SEC-HPLC and non-reducing CE-SDS methods were significant (SEC-HPLC reflects purity changes caused by protein aggregation, while non-reducing CE-SDS reflects purity changes caused by protein fragmentation). In the charge variants of each sample, the principal components decreased significantly, mainly due to the deamidation of antibody A. The purity and charge variant results indicate that antibody A exhibits superior stability under pH conditions of 5.0-6.0.

[0296] Example 2: Prescription Screening Study

[0297] 1. Experimental Design

[0298] This experiment mainly investigated the effect of excipients on the stability of antibody A. Detailed formulation information is shown in Table 3.

[0299] Table 3. Prescription Information Table

[0300] Solutions F1 through F6 were prepared as follows: All components of each formulation listed in Table 3, except for antibody A and polysorbate 80, were reconstituted with water for injection to form buffer solutions (for solutions F1, F2, F3, F4, and F6, the total histidine concentration of 10 mM was generated from 0.12 mg / mL histidine and 1.94 mg / mL histidine hydrochloride). Antibody A was then ultrafiltered and added to each formulation solution. After replacement, the protein concentration of each formulation was adjusted to 100 mg / mL (specific concentration values ​​are given at T0 in Table 8). For solution F5, the pH was adjusted to 5.2 with dilute hydrochloric acid. Polysorbate 80 was added to a final concentration of 0.3 mg / mL.

[0301] Stress stability experiments were conducted on liquids F1 to F6, including freeze-thaw stability studies. Detailed experimental conditions and sampling plans are shown in Table 4.

[0302] Table 4. Stability Testing Scheme Note: All samples taken at the above time points were first frozen at -40℃ and stored for testing. They were then thawed and sent for testing as needed.

[0303] 2. Experimental Results

[0304] After being placed at 40°C for 2 weeks, the liquid formulations F1 to F6 showed no difference in appearance, pH, and protein content compared to T0 (i.e., the time point before the start of the stability study). The experimental results are shown in Tables 5, 6, and 7.

[0305] Table 5: Appearance of liquid formulations from F1 to F6 Note: The standard for acceptable appearance is "clear to slightly opalescent, colorless to slightly yellow liquid, free of foreign matter".

[0306] Table 6: pH values ​​of liquid formulations F1 to F6

[0307] Table 7: Protein concentrations (mg / ml) of liquid formulations F1 to F6

[0308] However, after the liquid formulations of F1 to F6 were placed at 40°C for 2 weeks, the acidic and alkaline components changed significantly. The experimental results are shown in Table 8, and the trend of change is shown in Figure 2.

[0309] Table 8: Charge heterogeneity (%) of liquid formulations from F1 to F6

[0310] The experimental results in Table 8 and Figure 2 demonstrate that the liquid formulations F1 to F6 did not significantly improve the dramatic change in the acid-base peak caused by deamidation at the N325 site of antibody A.

[0311] Example 3: Prescription Screening Study

[0312] 1. Experimental Design

[0313] Solution formulations of F7 to F9 were prepared using the same method as described in Examples 2 for the liquid formulations of F1 to F6 (formulation information is shown in Table 9). More specifically, the liquid formulation of F7 (same as F2 in Example 2) was prepared as follows: 0.72 mg histidine, 11.64 mg histidine hydrochloride, 300.00 mg sucrose, and 120.00 mg mannitol were diluted to 6 mL with water for injection. 600.0 mg antibody A was added via UFDF ultrafiltration, the mixture was filtered sterilized, 1.8 mg polysorbate 80 was added, and the mixture was mixed thoroughly, filtered, and dispensed into vials. The vials were then partially capped, lyophilized using the parameters in Table 12, fully capped, and crimped.

[0314] Then, it was freeze-dried according to the freeze-drying process described in Example 4 to obtain freeze-dried formulations of F7 to F9.

[0315] Table 9. Prescription Information Table

[0316] Liquid formulations and lyophilized formulations of F7 to F9 were placed at 40℃±2℃, and freeze-thaw stability studies were conducted on the liquid formulations simultaneously (detailed experimental conditions and sampling plan are shown in Table 4).

[0317] The stability test results of the liquid formulations from F7 to F9 are shown in Table 10, and the trends are shown in Figure 3 (A, B, C).

[0318] Table 10. Results of stability studies of liquid formulations from F7 to F9 Note: (a) The standard for acceptable appearance is "clear to slightly opalescent, colorless to slightly yellow liquid, free of foreign matter"; (b) The standard for acceptable visible foreign matter is "free of foreign matter".

[0319] The results showed that after being stored at 40℃±2℃ for 2 weeks, the appearance, visible foreign matter, protein content, and pH of the liquid formulations F7, F8, and F9 did not change significantly. However, the purity detected by SEC-HPLC decreased by 1.5%, 1.5%, and 1.7%, respectively, while the purity detected by non-reducing CE-SDS decreased by 2.0%, 1.6%, and 2.1%, respectively. The charge variant—principal component—decreased significantly to 3.8%–3.9%. After three freeze-thaw cycles, the physicochemical properties of each formulation remained essentially unchanged. Among the three solution samples, F8 performed best.

[0320] The results of the stability studies of the freeze-dried formulations F7 to F9 are detailed in Table 11, and the trends are shown in Figure 3 (D, E, F).

[0321] Table 11. Results of stability studies of lyophilized formulations from F7 to F9 Note: (a) The standard for acceptable appearance is "clear to slightly opalescent, colorless to slightly yellow liquid, free of foreign matter"; (b) The standard for acceptable visible foreign matter is "free of foreign matter"; (c) *In the early stages of development, the reconstitution volume was not studied, resulting in a large difference in concentration after reconstitution with water for injection.

[0322] The results showed that after being stored at 40℃±2℃ for 2 weeks, the appearance, visible foreign matter, and pH value of the lyophilized formulations F7, F8, and F9 did not change significantly. The purity of F7, F8, and F9, as determined by SEC-HPLC, decreased by 0.8%, 0.8%, and 2.8%, respectively; the purity determined by non-reducing CE-SDS decreased by 1.4%, 1.6%, and 3.4%, respectively; and the charge variant-principal component decreased by 1.8%, 2.0%, and 3.4%, respectively, remaining essentially stable. In the stability study of the lyophilized samples, F7 and F8 showed better performance. The protein content of F7 to F9 varied to some extent, mainly due to the smaller sample size and the lack of investigation into the effect of reconstitution volume on protein content detection. To more accurately determine the protein content, the stability of the lyophilized formulation of F8 was further investigated in Example 4.

[0323] Example 4: Prescription Determination Study

[0324] 1. Experimental Design

[0325] This experiment aims to further confirm the stability of the lyophilized formulation F8.

[0326] The liquid formulation of F8 was prepared as described in Example 3. Lyophilization was performed using the parameters in Table 12. The resulting samples were then subjected to stability studies under the conditions given in Table 13.

[0327] Table 12. Freeze-drying process parameters

[0328] Table 13. Stability Testing Scheme

[0329] 2. Experimental Results

[0330] The results of the stability study of the freeze-dried formulation of F8 are detailed in Table 14.

[0331] Table 14. Stability study results of the lyophilized formulation of F8 Note: (a) The standard for acceptable appearance is "clear to slightly opalescent, colorless to slightly yellow liquid, free of foreign matter"; (b) The standard for acceptable visible foreign matter is "free of foreign matter".

[0332] The results in Table 14 show that the appearance and visible foreign matter of the samples at all time points were acceptable, and there were no significant changes in protein content and pH value. After being placed at 40℃ for 4 weeks, the SEC main peak decreased by 1.2%, and there were no significant changes in the components of the charge variant; the content of polysorbate 80 did not change significantly; and there were no significant changes in biological activity.

[0333] Based on the results of Examples 1-4, it is evident that the pH value of the solution affects the stability of antibody A. A pH of 5.0-6.0 in the solution preparation before freeze-drying is more conducive to the stability of antibody A. The solution preparation must be stored at low temperatures, while the freeze-dried preparation exhibits significantly improved stability and can be stored at room temperature or even higher temperatures. Long-term stability tests show that the freeze-dried preparation can be stably stored for at least 36 months under refrigerated conditions (2°C to 8°C).

[0334] Example 5. Preparation and property verification of antibody A

[0335] I. Preparation of Antibody A

[0336] For the preparation and property verification of antibody A, please refer to the preparation and property verification of Hz33B9C5 in PCT / CN2024 / 090122.

[0337] The sequence of antibody A was cloned into the pcDNA3.1 vector for later use. ExpiCHO cells were harvested, and the cell density was measured to be 8.0–10 x 10⁻⁶ cells / cm². 6The viability of cells should be between 95-99%, and the cell density should be adjusted to 6.0 x 10⁶ cells / mL. 6 Viable cells / mL. Take an appropriate-sized container and add Fectamine (source: Gibco) transfection buffer at 8% of the cell volume. Add the corresponding amount of plasmid at 0.8 μg / mL for cell transfection. Filter the transfection buffer containing DNA plasmid through a 0.22 μm filter to sterilize it into a new clean container. Add FectPro (source: Polyplus) transfection reagent at a ratio of 3.2 μL / mL to the filtered mixture and let it stand for 10 min. Quickly add the complex formed by the transfection reagent and plasmid DNA to the cells, gently shaking the flask while adding. Incubate at 36.5℃ on a shaker with 8% CO2. After 18-22 h of culture, add Enhancer (source: Gibco) at 6 μL / mL of cell volume and Feed (source: Gibco) at 300 μL / mL of cell volume. On day 7, collect the cell supernatant. Purify the protein using affinity chromatography to obtain the final antibody molecule. The purified antibody solution was centrifuged in a 15 ml ultrafiltration centrifuge tube at 4000 rpm for 10 minutes. The protein was then diluted with PBS and centrifuged again at 4000 rpm for 10 minutes. This process was repeated several times, changing the buffer solution each time. The antibodies were pooled after each buffer change, and the antibody concentration was measured. The components of the monoclonal antibody were further qualitatively analyzed using liquid chromatography-mass spectrometry (LC-MS) and size exclusion chromatography (SEC), and quantified using a UV-Vis spectrophotometer.

[0338] II. In vivo activity validation of antibody A – Data in an experimental autoimmune encephalomyelitis (EAE) mouse model. Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that leads to encephalitis and demyelination. MS is considered an autoimmune disease caused by autoreactive T cells, with symptoms including muscle rigidity and paralysis, visual disturbances and blindness, sensory loss, and ataxia, characterized by recurrent episodes. Several distinct animal models of MS exist, among which the experimental autoimmune encephalomyelitis (EAE) model is widely used in MS research due to its similar inflammatory and demyelinating pathological features to MS. The EAE model is induced by immunotherapy with myelin proteins (in this experiment, the myelin oligodendrocyte glycoprotein MOG) or peptides of these proteins. This is likely due to the peripheral activation of myelin-specific T cells, which cross the blood-brain barrier into the central nervous system and are reactivated, triggering a series of inflammatory responses leading to demyelination and axonal cell apoptosis, ultimately resulting in nerve damage and loss of function. A standardized scoring system is used to assess clinical symptoms, which measures the degree of disease induction.

[0339] The scoring criteria for the experimental autoimmune encephalomyelitis (EAE) mouse model used in this experiment are as follows:

[0340] 0 points: Mice are healthy

[0341] 1 point: The mouse's tail was weak.

[0342] 2 points: One hind limb of the mouse is paralyzed.

[0343] 3 points: The mouse's two hind limbs were paralyzed.

[0344] 4 points: Mouse weight loss of 20% or more

[0345] 5 points: The mouse is dead or near death.

[0346] Furthermore, measuring mouse body weight can indirectly indicate the health status of the mice, and detecting the titer of anti-MOG antibodies in mouse peripheral blood serum reflects the inhibitory effect of the antibody of this invention on antibody production in mice. Figures 6-8 show the experimental results.

[0347] The details of the method are as follows:

[0348] a) Reagent preparation:

[0349] 1. Inactivated tuberculosis stock solution (H37Ra stock solution) (100mg / ml): 10ml of complete Freund's adjuvant + 100mg H37Ra dissolved by sonication;

[0350] 2. MOG35-55 stock solution (10mg / ml): 5ml complete Freund's adjuvant + 50mg MOG35-55;

[0351] 3. Pertussis virus stock solution (PTX stock solution) (100ug / ml): 50ug PTX + 500ul DPBS (aliported and frozen at -40 degrees Celsius);

[0352] 4. Working solution for inactivated Mycobacterium tuberculosis (H37Ra): 500ul of complete Freund's adjuvant + 500ul of stock solution for inactivated Mycobacterium tuberculosis;

[0353] 5. MOG35-55 emulsifier: 2ml MOG35-55 emulsifier = 200ul MOG35-55 stock solution + 800ul DPBS + 1ml H37Ra working solution. Use after thorough emulsification.

[0354] 6. Pertussis virus working solution (PTX working solution): 30ul PTX stock solution + 1970ul DPBS (prepare fresh before use)

[0355] b) Model approach:

[0356] 1. Five 8-10 week old mice, all female, were administered control (PBS), hIgG, INX-021, antibody A (Hz33B9C5), and Hz47E2 to each group, respectively, at a dose of 10 mg / kg.

[0357] 2. Day 0: 100 μL of MOG35-55 emulsion was injected into each of the two points on the back, and 200 μL of PTX working solution was injected into the tail vein. Antibody or control was administered via intraperitoneal injection.

[0358] 3. Day 3: PTX working solution was injected via tail vein, and antibody or control was administered via intravenous injection.

[0359] 3. On Day 7 and Day 10, administer antibody or control via intraperitoneal injection (IP).

[0360] 4. During Days 0, 3, 7, 10, and 12-21, the severity of the disease was evaluated using a 0-5 point system, and the weight of the mice was recorded.

[0361] 5. On Day 7, Day 14, and Day 21, serum was collected by taking blood from the orbital cavity of mice, and the titer of Anti-MOG antibody in the serum was detected by ELISA.

[0362] Equipment: Thermo Fisher Multiskan-FC

[0363] Table 15: Reagents and Raw Materials

[0364] As shown in Figures 6-7, administration of antibody A (Hz33B9C5) of the present invention at 10 mg / kg significantly reduced the clinical score of experimental autoimmune encephalomyelitis (EAE) without affecting the body weight of mice. These effects were similar to those of the uninduced MOG control group and superior to the positive control INX-021. These results indicate that antibody A (Hz33B9C5) has the ability to improve experimental autoimmune encephalomyelitis (EAE) and autoimmune diseases such as multiple sclerosis (MS).

[0365] As shown in Figure 8, after administering antibody A (Hz33B9C5) at a dose of 10 mg / kg, especially after 21 days of immunization, the antibody of the present invention showed a lower titer of Anti-MOG antibody compared to INX-021. This indicates that the antibody of the present invention has a stronger inhibitory effect on humoral immunity compared to INX-021, and preferably a stronger inhibitory effect on antibody production.

[0366] III. In vivo activity verification of antibody A – inhibition of antihemocyanin antibody production in mice

[0367] Because peptides, small proteins, and some drug molecules are haptens and generally lack immunogenicity, they require carrier proteins such as hemocyanin to stimulate the immune system to produce antibodies. Therefore, hemocyanin (KLH) is widely used in research, biotechnology, and therapeutic applications. Furthermore, since hemocyanin originates from limpets (gastropods), its growth and development differ significantly from that of mammals, resulting in a lower likelihood of false positives in some mammalian-based immunological studies.

[0368] After injecting the antibody of this invention into humanized CD40 / CD40L mice, the antibody A inhibited the interaction between CD40L and CD40, thereby suppressing the maturation and differentiation of B cells and the production of anti-hemocyanin antibodies. Figure 9 shows the experimental results.

[0369] The details of the method are as follows:

[0370] a) Reagent preparation: Based on 250ug of hemocyanin per mouse, dissolve the corresponding amount in 100ul of PBS for later use;

[0371] b) Model approach:

[0372] 1. Five mice were used in each group, with an age of about 8-10 weeks. They were administered control hIgG, INX-021 (3mpk), and antibody A (Hz33B9C5) (0.3mpk, 1mpk, and 3mpk), respectively.

[0373] 2. Day 0: Mix hemocyanin stock solution and alum adjuvant at a volume ratio of 1:1, inject intraperitoneally, and simultaneously administer antibody drugs to mice via IP.

[0374] 2. Day 7: Inject antibody drugs into mice via intraperitoneal injection (IP);

[0375] 3. On Day 7, Day 14, and Day 21, serum was collected by taking blood from the orbital cavity of mice, and the titer of Anti-MOG antibody in the serum was detected by ELISA.

[0376] Equipment: Thermo Fisher Multiskan-FC

[0377] Table 16: Reagents and Raw Materials

[0378] As shown in Figure 9, in humanized CD40 / CD40L mice, compared to INX-021, the concentration of anti-KLH IgG in the serum of mice treated with antibody A (Hz33B9C5) of this invention was lower. This indicates that the antibody of this invention can more strongly inhibit the interaction between CD40L and CD40, thereby inhibiting B cell maturation and differentiation via the T cell-dependent pathway, and ultimately reducing the production of anti-hemocyanin antibodies. Furthermore, in different antibody A (Hz33B9C5) dosage groups, the titer of anti-KLH IgG in mouse serum showed a dose-response trend, indicating that the efficacy of antibody A (Hz33B9C5) in this animal model is dose-dependent.

[0379] IV. Pharmacokinetics of Antibody A in Mice

[0380] Experimental materials

[0381] Reagent and Consumable Information

[0382] Instrument and equipment information

[0383] Experimental preparation

[0384] (1) 1×PBS buffer

[0385] Add 100 mL of 10×PBS buffer to a final volume of 1 L and mix well.

[0386] (2) Coating fluid

[0387] Take one packet of carbonate powder, dissolve it in 400mL of ultrapure water, and bring the volume up to 500mL. Mix well.

[0388] (3) Washing solution

[0389] Add 1 mL of Tween-20 to 1999 mL of 1×PBS buffer and mix well.

[0390] (4) Sealing liquid

[0391] Weigh 5g of skim milk powder, dissolve it in 100mL of washing solution, and mix well.

[0392] (5) Sample diluent (antibody diluent)

[0393] Weigh 1g of BSA, dissolve it in 100mL of washing solution, and mix well.

[0394] Experimental process

[0395] (1) Pre-coating: Coat the 96-well microplate one day in advance, and dilute the CD40L protein to 1 μg / mL with coating buffer. 100 μL per well, seal the plate with sealing membrane, and incubate at 4°C overnight.

[0396] (2) Washing the plate: Discard the pre-coating solution in the 96-well microplate, pat dry on absorbent paper, then add 300 μL of washing buffer to each well, shake to mix for 10 seconds, pat dry the washing buffer, and repeat the washing 3 times.

[0397] (3) Sealing: Add sealing liquid to each well using a pipette, 200 μL, seal with a sealing membrane, and incubate overnight at 4°C or for 2 hours at room temperature.

[0398] (4) Wash the plate: Repeat (2).

[0399] (5) Sample addition: Add 100 μL of diluted standard (antibody A (Hz33B9C5) and Hz47E2, concentrations of 1-512 ng / ml, 10 concentrations, 2-fold dilution), quality control samples (antibody A (Hz33B9C5) and Hz47E2, 5.12-200 ng / ml, 5 concentrations, 2-fold dilution) and test samples (serum obtained after blood collection from mouse orbital sinuses at a certain time) to each well and incubate at room temperature for 2 hours, taking care to avoid light.

[0400] (6) Washing the plate: Discard the solution in the 96-well microplate, pat dry on absorbent paper, then add 300 μL of washing buffer to each well, shake to mix for 10 seconds, pat dry the washing buffer, and repeat the washing 6 times.

[0401] (7) Add enzyme-labeled antibody for incubation: Add diluted Goat anti-Human IgG Fc-HRP (25ng / mL) 100μL per well and incubate at room temperature for 1 hour, taking care to avoid light.

[0402] (8) Wash the plate: Repeat 3.1.6.

[0403] (9) Color development: Add 100 μL of TMB substrate to each well of a 96-well microplate and develop the color for 5-10 minutes at room temperature in the dark.

[0404] (10) Termination: Add 50 μL of ELISA termination solution to each well, shake at medium speed for 10 seconds, and read the OD450nm / 620nm values ​​within 30 minutes.

[0405] Results Analysis

[0406] (1) The concentration measurement value is retained to 4 decimal places, and the average concentration value is retained to 2 decimal places. Mean is used to indicate the concentration.

[0407] (2) The absorbance values ​​at 450nm and 620nm are provided by the microplate reader and cannot be changed. OD of sample = (A450 - A620) / sample. Absorbance values ​​are retained to four decimal places. Each sample is loaded in duplicate. Accuracy (used to evaluate the relative error relative to the theoretical value, abbreviated as %RE) and precision (used to evaluate the relative standard deviation, abbreviated as %CV) are retained to one decimal place. The calculation formulas are as follows:

[0408] Precision (%CV) = Standard Deviation (SD) / Average Measured Concentration × 100%

[0409] Accuracy (%RE) = (Average measured concentration - Theoretical concentration) / Theoretical concentration × 100%

[0410] (3) Experimental data: The concentration values ​​were obtained by fitting a four-parameter Logistic curve using Thermo's Skanit 3.1 analysis software. The average concentration, accuracy, and precision were calculated using the built-in calculation function of Excel.

[0411] The equation of the correction curve is: y = d + (ad) / (1 + (x / c)^b)

[0412] As shown in Table 17 below, antibody A (Hz33B9C5) has a long in vivo half-life, which indicates that the antibody of the present invention improves the pharmacokinetics of the anti-CD40L antibody and has excellent pharmacokinetics.

[0413] Table 17. Pharmacokinetics of the anti-CD40L antibody of the present invention

[0414] The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0415] Sequence information:

Claims

1. A pharmaceutical formulation comprising an anti-CD40L antibody or an antigen-binding fragment thereof, a pH adjuster, a protein protectant, and a surfactant, wherein the formulation is a lyophilized formulation or a liquid formulation, and the liquid formulation has a pH of 5.0-6.0, preferably a pH of 5.0-5.5, more preferably a pH of about 5.2, wherein the anti-CD40L antibody or its antigen-binding fragment comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), and a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or respectively composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:

9. The amino acid sequences shown in NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 are composed of amino acid sequences.

2. The pharmaceutical formulation according to claim 1, wherein the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:10 or the nucleic acid sequence shown in SEQ ID NO:20 ... The amino acid sequence encoded by the amino acid sequence shown in NO:10 or the nucleic acid sequence shown in SEQ ID NO:20, or the amino acid sequence composed of said sequences.

3. The pharmaceutical formulation according to any one of claims 1-2, wherein the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence encoded by the amino acid sequence shown in SEQ ID NO:4 or the nucleic acid sequence shown in SEQ ID NO:19 or is composed of the amino acid sequence, and the light chain variable region comprises an amino acid sequence encoded by the amino acid sequence shown in SEQ ID NO:10 or the nucleic acid sequence shown in SEQ ID NO:20 or is composed of the amino acid sequence.

4. The pharmaceutical formulation according to any one of claims 1-3, wherein the anti-CD40L antibody or its antigen-binding fragment further comprises an Fc region, for example, wherein the Fc region is or is derived from human IgG Fc, for example, is or is derived from human IgG1 Fc, is or is derived from human IgG2 Fc, is or is derived from human IgG3 Fc or is derived from human IgG4 Fc; preferably, the Fc region comprises a mutation that reduces or eliminates effector function, and / or eliminates the binding affinity to FcγR while retaining the binding affinity to FcRn, preferably, the mutation is an L234A / L235A and / or P329 deletion mutation, preferably, the Fc region (i) Contains or consists of the amino acid sequence shown in SEQ ID NO:13 or 14; (ii) Contains an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:13, for example, 95%, 96%, 97%, 99% or higher identity; or (iii) Contains an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:14, such as 95%, 96%, 97%, 99% or higher, and contains L234A / L235A and P329 deletion mutations.

5. The pharmaceutical formulation according to any one of claims 1-4, wherein the anti-CD40L antibody or its antigen-binding fragment further comprises a heavy chain constant region, said heavy chain constant region being or derived from a constant region of (human) IgG1, IgG2, IgG3 or IgG4, preferably, said heavy chain constant region (i) Contains or is composed of an amino acid sequence selected from or represented by SEQ ID NO:5 or 16; (ii) comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:16; or (iii) Contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:5, and has L234A / L235A and P329 deletion mutations. and / or The anti-CD40L antibody or its antigen-binding fragment further comprises a light chain constant region, which is or is derived from the light chain constant region of (human) lambda or Kappa. Preferably, the light chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:11; or comprises or is composed of the amino acid sequence shown in SEQ ID NO:

11.

6. The pharmaceutical formulation according to any one of claims 1-5, wherein the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain, wherein the heavy chain (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:6; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

6. or The anti-CD40L antibody or its antigen-binding fragment comprises a light chain, wherein the light chain (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

12.

7. The pharmaceutical formulation according to any one of claims 1-6, wherein the anti-CD40L antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:6, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:

12.

8. The pharmaceutical formulation according to any one of claims 1-7, wherein the anti-CD40L antibody is a humanized antibody or a chimeric antibody.

9. The pharmaceutical formulation according to any one of claims 1-8, wherein the antigen-binding fragment of the anti-CD40L antibody is selected from the following antibody fragments: Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibody, single-chain antibody (e.g., scFv); single-domain antibody (sdAb) such as VHH, bivalent antibody or fragment thereof, or camelid antibody or diabody antibody.

10. The pharmaceutical formulation according to any one of claims 1-9, wherein the anti-CD40L antibody comprises two heavy chains and two light chains, wherein the heavy chains comprise the amino acid sequence shown in SEQ ID NO:6 and the light chains comprise the amino acid sequence shown in SEQ ID NO:12, preferably the amino acid sequence of the anti-CD40L antibody is shown in Figure 5.

11. The pharmaceutical formulation according to any one of claims 1-10, wherein the formulation is a liquid formulation and the anti-CD40L antibody or its antigen-binding fragment is present in the liquid formulation at a concentration of 50-100 mg / mL, for example about 50, 60, 70, 80, 90 or 100 mg / mL, or the formulation is a lyophilized formulation and the anti-CD40L antibody or its antigen-binding fragment constitutes 30-90% w / w, preferably 40-80% w / w, more preferably 50-60% w / w, for example about 58% w / w, of the lyophilized formulation.

12. The pharmaceutical formulation according to any one of claims 1-11, wherein the formulation comprises 100 mg-1000 mg, for example 180 mg-550 mg, 200 mg-540 mg, 300 mg-600 mg, 300 mg-720 mg, 500 mg-1000 mg or 500 mg-800 mg, for example about 180 mg, 300 mg, 540 mg or 600 mg of the anti-CD40L antibody or its antigen-binding fragment.

13. The pharmaceutical preparation according to any one of claims 1-12, wherein The pH adjuster is selected from one or more of the following: acetate (e.g., sodium acetate, potassium acetate), carbonate (e.g., sodium carbonate, sodium carbonate), bicarbonate (e.g., sodium bicarbonate), citrate (e.g., sodium citrate), amino acids (e.g., histidine, glycine, lysine, and arginine), amino acid salts (e.g., amino acid hydrochloride or sodium amino acid salts, such as histidine hydrochloride, arginine hydrochloride, monosodium glutamate), phosphates (e.g., sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate), tris(hydroxymethyl)aminomethane (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), acetic acid, carbonic acid, citric acid, phosphoric acid, hydrochloric acid, and sodium hydroxide. or The pH adjuster is selected from one or more of sodium acetate, amino acids (e.g., histidine, glycine, lysine, and arginine), amino acid salts (e.g., amino acid hydrochloride or sodium amino acid salts, such as histidine hydrochloride, arginine hydrochloride), phosphates (e.g., sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate), tris(hydroxymethyl)aminomethane (Tris), and hydrochloric acid. or The pH adjuster is selected from one or more of sodium acetate, amino acids (e.g., histidine, glycine, lysine, arginine, and methionine), amino acid hydrochlorides (e.g., histidine hydrochloride, arginine hydrochloride), and hydrochloric acid. or The pH adjuster is selected from one or more of sodium acetate, hydrochloric acid, histidine, and histidine hydrochloride. or The pH adjuster is histidine and histidine hydrochloride. Preferably, the pH adjuster is histidine and histidine hydrochloride in a weight ratio of 1:10 to 1:22, more preferably 1:12 to 1:20, more preferably 1:14 to 1:18, for example about 1:

16.

14. The pharmaceutical preparation according to any one of claims 1-13, wherein, The pharmaceutical preparation is a liquid preparation, the pH adjuster is histidine and histidine hydrochloride in a weight ratio of about 1:16, and the total concentration of histidine in the liquid preparation is about 10 mM, preferably the total concentration of histidine of about 10 mM consists of about 0.12 mg / mL free histidine and about 1.94 mg / mL histidine hydrochloride; or The pharmaceutical formulation is a lyophilized formulation, and the pH adjuster is histidine and histidine hydrochloride in a weight ratio of 1:10 to 1:22, preferably 1:12 to 1:20, more preferably 1:14 to 1:18, for example about 1:16, and the pH adjuster accounts for 0.5-3% w / w, for example 1-2% w / w, preferably about 1.2% w / w, of the lyophilized formulation.

15. The pharmaceutical preparation according to any one of claims 1-14, wherein The protein protectant is selected from one or more of the following substances: (1) Sugars, such as sucrose, lactose, trehalose, maltose, fructose, inulin oligosaccharides, glucan, glucose, maltodextrin, cyclodextrin, and dextran; (2) Polyols, such as sorbitol, xylitol, glycerol, and ethylene glycol; (3) Proteins, such as bovine serum albumin, human serum albumin, casein, and mucopolysaccharide proteins; (4) Cellulose, such as carboxymethyl cellulose, or The protein protectant is selected from one or more of the following substances: (1) Sugars, such as sucrose, lactose, trehalose, maltose, fructose, inulin oligosaccharides, dextran, and maltodextrin; (2) Polyols, such as sorbitol and xylitol, or The protein protectant is selected from one or more of the following substances: (1) Sugars, such as sucrose and trehalose; (2) Polyols, such as sorbitol, or The protein protectant is selected from one or more of sucrose, trehalose, and sorbitol; or The protein protectant is sucrose.

16. The pharmaceutical preparation according to any one of claims 1-15, wherein, The formulation is a liquid formulation, wherein the protein protectant comprises 1-15% w / w, preferably 2-10% w / w, more preferably 6-8% w / w, for example about 6.67% w / w; or the formulation is a liquid formulation, wherein the protein protectant is present in the liquid formulation at a concentration of 15-90 mg / mL, preferably 40-90 mg / mL, 50-80 mg / mL, more preferably about 50 mg / mL; or The formulation is a lyophilized formulation, wherein the protein protectant accounts for 10-90% w / w of the lyophilized formulation, preferably 20-80% w / w, more preferably 30-50% w / w, for example about 40.61% w / w.

17. The pharmaceutical preparation according to any one of claims 1-16, wherein, The surfactant may be a nonionic surfactant or an ionic surfactant. The nonionic surfactant may be selected from: sorbitan fatty acid esters, including polysorbates (e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85) and sorbitan esters (e.g., Span-20, Span-40, Span-60, Span-65, Span-80, Span-83, Span-85); polypropylene glycol ethylene oxide adducts, including poloxamer derivatives (e.g., poloxamer 188); and polyoxyethylene alkyl ethers, including benzyl ethers (Brij) (e.g., Brij). -30, Brij-35, Brij-52, Brij-56, Brij-58, Brij-76, Brij-28, Brij-92, Brij-96, Brij-98), wherein the ionic surfactant can be a cationic surfactant (e.g., dodecyltrimethylammonium chloride, octadecyltrimethylammonium chloride), anionic surfactant (e.g., sodium dodecylbenzenesulfonate, sodium dodecyl sulfonate), or amphoteric surfactant (e.g., methyl dodecylaminopropionate, sodium dodecylaminopropionate, sodium dodecylaminodiacetate, sodium octadecylaminopropionate, phosphatidylserine, lecithin, soybean lecithin), or The surfactant is a nonionic surfactant selected from: sorbitan fatty acid esters, including polysorbates (e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85) and sorbitan esters (e.g., Span-20, Span-40, Span-60, Span-65, Span-80, Span-83, Span-85); polypropylene glycol ethylene oxide adducts, including poloxamer derivatives (e.g., poloxamer 188); and polyoxyethylene alkyl ethers, including benzyl ethers (Brij) (e.g., Brij-30, Brij-35, Brij-52, Brij-56, Brij-58, Brij-76, Brij-28, Brij-92, Brij-96, Brij-98). or The surfactant is a nonionic surfactant selected from sorbitan fatty acid esters, such as polysorbates (e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85) and sorbitan esters (e.g., Span-20, Span-40, Span-60, Span-65, Span-80, Span-83, Span-85). or The surfactant is polysorbate, preferably polysorbate-80.

18. The pharmaceutical preparation according to any one of claims 1-17, wherein, The formulation is a liquid formulation, wherein the surfactant accounts for 0.001-0.1% w / w, preferably 0.01-0.05% w / w, for example about 0.03% w / w; or the formulation is a liquid formulation, wherein the surfactant is present in the liquid formulation at a concentration of 0.1 mg / mL to 0.5 mg / mL, preferably 0.2 mg / mL to 0.4 mg / mL, more preferably about 0.3 mg / mL. or The formulation is a lyophilized formulation, and the surfactant accounts for 0.04-0.7% w / w of the lyophilized formulation, preferably 0.1-0.4% w / w, more preferably 0.1-0.3% w / w, for example about 0.17% w / w.

19. The pharmaceutical preparation according to claim 1, wherein, The formulation is a liquid formulation comprising (1) an anti-CD40L antibody or its antigen-binding fragment (e.g., an anti-CD40L monoclonal antibody, preferably antibody A), wherein the anti-CD40L antibody or its antigen-binding fragment is preferably present in the liquid formulation at a concentration of 50-100 mg / mL, for example, about 50, 60, 70, 80, 90 or 100 mg / mL; (2) a protein protectant, wherein the protein protectant is preferably sucrose, and is preferably present in the liquid formulation at a concentration of 15-90 mg / mL, for example, 40-90 mg / mL, 50-80 mg / mL, more preferably about 50 mg / mL; and (3) a surfactant, wherein the surfactant is preferably polysorbate (e.g., polysorbate 80), and is preferably present in the liquid formulation at a concentration of 0.05-1 mg / mL. More preferably, it is present at a concentration of 0.1-0.5 mg / mL, for example, about 0.3 mg / mL; (4) a pH adjuster, preferably selected from histidine and its pharmaceutically acceptable salts (e.g., histidine hydrochloride), the amount of which causes the liquid formulation to have a pH of 5.0-6.0, preferably 5.0-5.5, more preferably about 5.2; (5) an aqueous solvent for injection, selected from 0.9% sodium chloride injection, 5% glucose injection and water for injection, preferably water for injection; and optionally also includes (6) a pharmaceutically acceptable excipient, preferably mannitol, present in the liquid formulation at a concentration of 10 mg / mL to 30 mg / mL, preferably 15 mg / mL to 25 mg / mL, more preferably about 20 mg / mL; or The formulation is a lyophilized formulation comprising (1) 50-100 parts by weight, for example about 50, 60, 70, 80, 90 or 100 parts by weight of an anti-CD40L antibody or its antigen-binding fragment (e.g., an anti-CD40L monoclonal antibody, preferably antibody A); (2) 15-90 parts by weight, for example 40-90 parts by weight, 50-80 parts by weight, more preferably about 50 parts by weight of a protein protectant (preferably sucrose); and (3) 0.05-1 parts by weight, more preferably 0.1-0.5 parts by weight, for example about 0. 3 parts by weight of a surfactant (preferably polysorbate, such as polysorbate 80); (4) 0.77-2.33 parts by weight, preferably 1.24-1.86 parts by weight, more preferably about 1.55 parts by weight, based on histidine, said pH adjuster being selected from histidine and pharmaceutically acceptable salts of histidine (e.g., histidine hydrochloride); and optionally also comprising (6) 10-30 parts by weight, preferably 15-25 parts by weight, more preferably about 20 parts by weight of a pharmaceutically acceptable excipient (preferably mannitol).

20. The pharmaceutical preparation according to claim 1, wherein The formulation is a liquid formulation containing approximately 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), approximately 10 mM histidine, approximately 50 mg / mL sucrose, approximately 20 mg / mL mannitol, approximately 0.3 mg / mL polysorbate 80, and a pH of approximately 5.

2. or The formulation is a liquid formulation containing approximately 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), approximately 10 mM histidine, approximately 80 mg / mL sucrose, approximately 0.3 mg / mL polysorbate 80, and a pH of approximately 5.

2. or The formulation is a lyophilized formulation containing about 100 parts by weight of anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 parts by weight of histidine, about 1.94 parts by weight of histidine hydrochloride, about 50 parts by weight of sucrose, about 20 parts by weight of mannitol, and about 0.3 parts by weight of polysorbate 80. or The formulation is a lyophilized formulation containing about 100 parts by weight of anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 parts by weight of histidine, about 1.94 parts by weight of histidine hydrochloride, about 80 parts by weight of sucrose, and about 0.3 parts by weight of polysorbate 80. or The formulation is a liquid formulation containing approximately 100 mg / mL of anti-CD40L monoclonal antibody (preferably antibody A), approximately 10 mM histidine, approximately 20 mg / mL sucrose, approximately 40 mg / mL mannitol, approximately 0.3 mg / mL polysorbate 80, and a pH of approximately 5.

2. or The formulation is a lyophilized formulation containing about 100 parts by weight of anti-CD40L monoclonal antibody (preferably antibody A), about 0.12 parts by weight of histidine, about 1.94 parts by weight of histidine hydrochloride, about 20 parts by weight of sucrose, about 40 parts by weight of mannitol, and about 0.3 parts by weight of polysorbate 80.

21. The pharmaceutical formulation of claim 20, wherein the formulation is a liquid formulation, and the approximately 10 mM histidine comprises approximately 0.12 mg / mL histidine and approximately 1.94 mg / mL histidine hydrochloride.

22. The pharmaceutical preparation according to any one of claims 1-21, wherein the preparation contains about 600 mg of antibody.

23. A pillbox comprising a pharmaceutical preparation according to any one of claims 1-22 and instructions for using the preparation to prevent or treat an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway.

24. The kit of claim 23, wherein the kit comprises a pharmaceutical preparation of any one of claims 1-22 and instructions for using the preparation to prevent or treat an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway, the preparation being a lyophilized preparation, and the kit further comprising a reconstituted solvent in a separate container, preferably selected from 0.9% sodium chloride injection, 5% glucose injection, and water for injection.

25. A pre-filled syringe containing a pharmaceutical formulation according to any one of claims 1-22.

26. A method for preparing a pharmaceutical formulation according to any one of claims 1-22, the method comprising the following steps: (1) Dissolve the pH adjuster and protein protectant in an injectable solvent, add the anti-CD40L antibody or its antigen-binding fragment, and optionally adjust the pH value to pH 5.0-6.0; (2) Add a surfactant to obtain the liquid formulation; Optionally, (3) the liquid formulation obtained in step (2) is freeze-dried to obtain the freeze-dried formulation.

27. The method according to claim 26, wherein the freeze-drying in step (3) comprises the following steps: (a) pre-freezing, for example, first pre-freezing at atmospheric pressure at -3°C to 7°C, preferably at 0°C to 4°C, more preferably at about 2°C for about 1 hour, and then pre-freezing at atmospheric pressure at -50°C to -40°C, preferably at -47°C to -43°C, more preferably at about -45°C for about 4 hours; (b) vacuuming, for example, vacuuming to about 32 Pa; (c) performing a first drying at vacuum at -25°C to -15°C, preferably at -22°C to -18°C, more preferably at about -20°C for about 36 hours; (d) performing a second drying at vacuum at 20°C to 30°C, preferably at 23°C to 27°C, more preferably at about 25°C for about 18 hours.

28. The method according to claim 26 or 27, wherein the method comprises steps (1) to (3), and further comprises step (4): (4) Purge the freeze-dried product from step (3) with nitrogen to 750-850 mbar and then plug it.

29. The pharmaceutical preparation according to any one of claims 1-22, for the prevention or treatment of diseases or conditions related to inappropriate activation of the CD40L / CD40-mediated pathway.

30. A method for preventing or treating an individual’s disease or condition related to inappropriate activation of the CD40L / CD40-mediated pathway, the method comprising injecting the individual with an effective amount of the pharmaceutical preparation of any one of claims 1-22.

31. The pharmaceutical formulation of claim 29 or the method of claim 30, wherein the disease or condition associated with inappropriate activation of the CD40L / CD40-mediated pathway is selected from: autoimmune diseases such as primary Sjögren's syndrome. Syndrome), systemic lupus erythematosus, multiple sclerosis, lupus nephritis, immune thrombocytopenic purpura (ITP), Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, psoriasis, myasthenia gravis, graft-versus-host disease, graft rejection and inflammatory bowel disease (IBD), neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), progressive bulbar palsy and spinal muscular atrophy, tumors, atherosclerosis, coronary heart disease, type 1 diabetes, asthma, allergic reactions and immune responses to recombinant drug products such as hemophilia factor VII.

32. The pharmaceutical formulation of claim 29 or the method of claim 30, wherein the disease or condition associated with inappropriate activation of the CD40L / CD40-mediated pathway is selected from autoimmune diseases, such as primary Sjögren's syndrome, systemic lupus erythematosus, multiple sclerosis, lupus nephritis, immune thrombocytopenic purpura (ITP), Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, psoriasis, myasthenia gravis, graft-versus-host disease, graft rejection, and inflammatory bowel disease (IBD).

33. The pharmaceutical formulation of claim 29 or the method of claim 30, wherein the disease or condition associated with inappropriate activation of the CD40L / CD40-mediated pathway is selected from autoimmune diseases, such as primary Sjögren's syndrome and systemic lupus erythematosus.

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