Solution formulation of humanized anti‑tumor necrosis factor superfamily member 4 (OX40L) antibody or antigen‑binding fragment thereof

Abstract

Provided is a solution formulation of a humanized anti‑OX40L antibody or an antigen‑binding fragment thereof. Also provided are preparation of the solution formulation, and a method and use thereof for preventing or treating a disease or disorder associated with abnormal activation of an OX40L / OX40-mediated pathway.

Description

Solution formulation of humanized anti-tumor necrosis factor superfamily member 4 (OX40L) antibody or its antigen-binding fragment

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202510127931.6, filed on February 4, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the pharmaceutical field. Specifically, it relates to solution formulations of humanized anti-tumor necrosis factor superfamily member 4 (OX40L) antibodies or their antigen-binding fragments, and related dosage forms obtained therefrom. This invention also relates to methods for preparing said solution formulations and their methods and uses for the prevention or treatment of autoimmune diseases or allogeneic transplantation. Background Technology

[0004] T-cell co-stimulatory molecule OX40 and its homologous ligand OX40L have attracted widespread research interest as therapeutic targets for T-cell-mediated diseases.

[0005] OX40 is a type I transmembrane glycoprotein composed of approximately 275 amino acids, featuring three complete cysteine-rich domains (CRDs) and a partially C-terminal CRD. The apparent molecular weight of OX40 is 50 kDa. OX40, along with other TNFRSF members (e.g., 4-1BB, CD27, CD30, and CD40), are T cell co-stimulatory molecules that function at different stages to regulate and control immune responses. Unlike other constitutive T cell co-stimulatory receptors (such as CD28 and CD27), OX40 is not expressed on naive T cells. OX40 is transiently induced by a signal following TCR binding after antigen (Ag) recognition, peaking at 48–72 h in activated T cells (including activated CD4+ and CD8+ T cells, as well as neutrophils, natural killer (NK) cells, and natural killer T cells (NKT)). The only exception is that OX40 is constitutively expressed in mouse FOXP3+ Treg cells. The kinetics of OX40 expression involve many factors, including the cytokine environment, antigen persistence, inflammatory environment, and the influence of other co-stimulatory pathways. OX40-deficient T cells may typically proliferate and differentiate into effector T cells 2–3 days after TCR recruitment, but they exhibit a significant decrease in survival after 12–13 days. That is, OX40 signaling may not affect the initial activation phase of T cells, but it may play an important role in maintaining late-stage proliferation and survival of T cells in the effector phase.

[0006] OX40 ligand (OX40L, also known as CD252, CD134L, or gp34) is also a member of the TNFR superfamily. OX40L is a type II glycoprotein with a 23-amino acid cytoplasmic tail and a 133-amino acid extracellular domain. Mouse OX40L can stimulate human and mouse T cells; however, human OX40L only stimulates human T cells. OX40L is primarily expressed on activated antigen-presenting cells (APCs) such as dendritic cells (DCs), activated B cells, and macrophages. The cell types that can be induced to express OX40L are much broader. In addition to APCs, OX40L is also expressed on hematopoietic cells such as activated NK cells, mast cells, or responsive CD4+ T cells, and non-hematopoietic cells such as endothelial cells or smooth muscle cells. (Yu Fu et al., The significance of OX40 and OX40L to T-cell biology and immune disease, Immunological Reviews 2009, Vol.229:173–191). OX40L interacts with OX40 and induces receptor trimerization, thereby activating the downstream NfKB signaling pathway, promoting the formation of memory T cells, the secretion of inflammatory factors, and inhibiting the production of regulatory T cells, thus playing a role in promoting cellular immune responses.

[0007] Autoimmune diseases are characterized by the production of reactive autoantibodies against their own antigens due to the immune system's failure to maintain self-tolerance. Because the pathogenesis is poorly understood, some well-known autoimmune diseases have a high incidence rate. These include experimental autoimmune encephalomyelitis (EAE), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), colitis, autoimmune experimental uveitis (AEU), type 1 diabetes, and multiple sclerosis (MS).

[0008] Because T cells play a crucial role in controlling the immune response and co-stimulatory effects of the OX40-OX40L interaction, targeting the OX40-OX40L interaction may improve autoantigen-specific T cell responses. Therefore, experiments have been conducted to verify whether targeting the OX40-OX40L interaction by blocking OX40 or OX40L has a therapeutic benefit for autoimmune diseases, including EAE, SLE, RA, colitis, AEU, type 1 diabetes, MS, graft-versus-host disease (GVHD), and inflammatory bowel disease (IBD). Studies have shown that OX40 is upregulated at autoimmune sites and is associated with disease severity. Therefore, inhibiting this signaling pathway has potential therapeutic effects on various immune-related diseases.

[0009] Given the large number of unmet clinical treatment needs, it is essential to develop drugs that target tumor necrosis factor superfamily member 4 (OX40L).

[0010] Drug stability is a crucial indicator of drug efficacy and safety. Obtaining formulations with good stability is key to maintaining the efficacy and safety of drugs during storage. However, protein formulations are prone to protein degradation and aggregation during formulation and storage, which can affect the safety and efficacy of drug products. The sensitivity of proteins requires mild conditions during formulation to avoid damaging their structure and function. Due to the diversity and complexity of proteins themselves, it is currently impossible to predict the formulation conditions required for optimizing antibody stability. This is especially true considering that different antibodies often have different CDR sequences, and these sequence differences lead to different stability properties of different antibodies 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. However, achieving a balance among the many requirements of antibody solution formulations is difficult, and there is an unmet need in the art for solution formulations of antibodies against humanized anti-tumor necrosis factor superfamily member 4 (OX40L) or its antigen-binding fragments.

[0011] The present invention aims to provide a solution formulation of an antibody against humanized anti-tumor necrosis factor superfamily member 4 (OX40L) or its antigen-binding fragment, which has suitable drug safety and efficacy for administration and is cost-effective, providing more options in clinical practice. Summary of the Invention

[0012] Invention Summary

[0013] This invention provides a solution formulation comprising an antibody against humanized anti-tumor necrosis factor superfamily member 4 (OX40L) or an antigen-binding fragment thereof, having a pH of about 5.0 to about 8.0. The inventors have found that solutions containing anti-OX40L antibodies or their antigen-binding fragments exhibit stability and efficacy suitable for parenteral administration, particularly injection, within a pH range of about 5.0 to about 8.0. The formulation of this invention is stable for at least 12 months, preferably more than 24 months, under storage conditions (2-8°C), without turbidity or visible foreign matter. pH shifts and changes in protein content, protein purity, and charge variants are within acceptable ranges, and the biological activity meets the standards.

[0014] The solution formulation of the present invention has good stability.

[0015] Therefore, in one aspect, a solution formulation comprising an anti-OX40L antibody or an antigen-binding fragment thereof is provided, having a pH of about 5.0 to about 8.0.

[0016] On the other hand, a pre-filled syringe containing the solution formulation of the present invention is provided.

[0017] In another aspect, solid formulations, such as freeze-dried or spray-dried formulations, obtained from the solution formulations of the present invention are provided.

[0018] On the other hand, the present invention provides formulations, such as the solution formulations of the present invention or the solid formulations obtained therefrom, for the prevention or treatment of diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway.

[0019] In another aspect, the use of the formulations of the present invention, such as the solution formulations of the present invention or the solid formulations obtained therefrom, is provided for the prevention or treatment of diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway.

[0020] In another aspect, the use of the formulations of the present invention, such as the solution formulations of the present invention or the solid formulations obtained therefrom, is provided in the preparation of medicaments for the prevention or treatment of diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway.

[0021] On the other hand, methods are provided for preventing or treating diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway in an individual, the methods comprising administering to the individual an effective amount of an formulation of the present invention, such as a solution formulation of the present invention or a solid formulation obtained therefrom.

[0022] On the other hand, a medicine box is provided that contains the formulation of the present invention, such as the solution formulation of the present invention or the solid formulation obtained therefrom, and instructions for use of said formulation for the prevention or treatment of diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway.

[0023] In another aspect, a method for preparing the solution formulation of the present invention is provided.

[0024] In another aspect, combinations of the formulations of the present invention, such as the solution formulations of the present invention or the solid formulations obtained therefrom, with one or more other therapies, such as pharmaceutical treatment and / or surgical treatment, are provided.

[0025] Various aspects, embodiments, features, and advantages of the present invention are described in detail below, and other aspects, embodiments, features, and advantages not specifically described herein can be determined by those skilled in the art based on the specification and the following claims.

[0026] Invention Details

[0027] In all aspects, embodiments, and examples of the present invention, the anti-OX40L antibody or its antigen-binding fragment comprises: three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO:4, and three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO:8, wherein the HCDR and LCDR can be determined according to any CDR determination scheme, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, the HCDR1 is determined according to the AbM scheme, and the HCDR2 and HCDR3 are determined according to the Kabat scheme, and the LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0028] In some embodiments, the anti-OX40L antibody or its antigen-binding fragment comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), 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 or consist of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0029] In some embodiments, the anti-OX40L 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 the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0030] In some embodiments, the anti-OX40L 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:8.

[0031] In some embodiments, the anti-OX40L 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 the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the anti-OX40L antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:8.

[0032] In some embodiments, the anti-OX40L antibody or its antigen-binding fragment further comprises an antibody heavy chain constant region. In some embodiments, the anti-OX40L antibody or its antigen-binding fragment further comprises an antibody light chain constant region. In some embodiments, the anti-OX40L antibody or its antigen-binding fragment further comprises both a heavy chain constant region and a light chain constant region.

[0033] In some embodiments, the antibody heavy chain constant region is a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4, preferably from human IgG1, IgG2, IgG3, or IgG4. More preferably, it is a heavy chain constant region of human IgG4.

[0034] In some preferred embodiments, the antibody heavy chain constant region

[0035] (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: 13; or

[0036] (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:13.

[0037] In some embodiments, the antibody heavy chain constant region contains a mutation that improves antibody stability, such as a mutation that eliminates IgG heterogeneity. In some embodiments, the mutation can help reduce the likelihood of antibody chain exchange in vivo. In some embodiments, the mutation is to change the serine residue at position 228 to proline. Therefore, in a preferred embodiment, the antibody heavy chain constant region has an S228P (EU number) mutation (e.g., referencing the human IgG4 heavy chain constant region number). In some embodiments, the antibody heavy chain constant region is the human IgG4 heavy chain constant region with S228P (EU number).

[0038] In some preferred embodiments, the antibody heavy chain constant region comprises 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, and has an S228P mutation. In some preferred embodiments, the antibody heavy chain constant region of the present invention comprises or is composed of the amino acid sequence shown in SEQ ID NO:11.

[0039] In some embodiments, the antibody light chain constant region is a light chain constant region derived from the lambda or kappa light chain constant region, preferably from the human lambda light chain constant region or the human kappa light chain constant region. In some embodiments, the antibody light chain constant region is a (human) kappa light chain constant region or a (human) lambda light chain constant region, more preferably a human kappa light chain constant region.

[0040] In some implementations, the antibody light chain constant region

[0041] (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 of SEQ ID NO: 12; or

[0042] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:12.

[0043] In some embodiments, the anti-OX40L antibody or its antigen-binding fragment further comprises an antibody heavy chain. In some embodiments, the anti-OX40L antibody or its antigen-binding fragment further comprises an antibody light chain. In some embodiments, the anti-OX40L antibody or its antigen-binding fragment further comprises a heavy chain and a light chain. In some embodiments, the antibody heavy chain comprises, or is composed of, the heavy chain variable region and the heavy chain constant region. In some embodiments, the antibody light chain comprises, or is composed of, the light chain variable region and the light chain constant region. In some embodiments, the antibody comprises two of the aforementioned heavy chains and two of the aforementioned light chains, or is composed of, two of the aforementioned heavy chains and two of the aforementioned light chains.

[0044] In some embodiments, 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:9; and / or 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:10. In some embodiments, the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:9, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:10.

[0045] In some embodiments, the anti-OX40L antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:9, and the light chain comprises the amino acid sequence shown in SEQ ID NO:10.

[0046] In some implementations, the anti-OX40L antibody is a monoclonal antibody.

[0047] In some implementations, the anti-OX40L antibody is humanized.

[0048] In one embodiment, the anti-OX40L antibody further encompasses its antibody fragments (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), bivalent antibodies, or linear antibodies.

[0049] In some embodiments, the anti-OX40L antibody also encompasses multispecific antibodies that specifically bind to OX40L, such as bispecific antibodies.

[0050] In some implementations, the anti-OX40L antibody is a full-length antibody.

[0051] In some embodiments, the anti-OX40L antibody or its antigen-binding fragment has one or more of the following properties:

[0052] a. The anti-OX40L antibody or its antigen-binding fragment can effectively block the interaction between OX40L and its receptor OX40 at the protein and cellular levels, such as binding and / or blocking OX40 signaling activation;

[0053] b. The antigen-binding fragment of the anti-OX40L antibody can effectively inhibit the activation, proliferation and / or differentiation of T cells, for example, by inhibiting the secretion level of T cells for inflammatory factors such as interleukins such as IL5 and / or IL3, or by inhibiting Th2 inflammatory responses;

[0054] c. The antigen-binding fragment of the anti-OX40L antibody can maintain the body weight (or maintain weight gain) of individuals with graft-versus-host disease, prevent clinical symptoms, reduce T cell activation, and / or inhibit the production of inflammatory factors (e.g., interferons such as IFNγ or interleukins such as IL-6); and / or

[0055] d. The probability of the antibody undergoing chain exchange in vivo is effectively reduced, thereby improving its stability.

[0056] In some specific embodiments, the anti-OX40L antibody or its antigen-binding fragment is derived from any anti-OX40L antibody or its antigen-binding fragment in PCT / CN2024 / 109523, the entire contents of which are incorporated herein by reference.

[0057] In the solution formulation of the present invention, the anti-OX40L antibody or its antigen-binding fragment may be present at a concentration of about 1-500 mg / mL, for example, about 10-400 mg / mL, about 20-300 mg / mL, about 40-200 mg / mL, about 50-150 mg / mL, or about 100-150 mg / mL. For example, the anti-OX40L antibody or its antigen-binding fragment may be present at concentrations of about 1, 20, 40, 60, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500 mg / mL or any combination thereof. Preferably, the anti-OX40L antibody or its antigen-binding fragment is present in the solution formulation at a concentration of about 100-150 mg / mL, for example, about 100 mg / mL or 150 mg / mL.

[0058] The solution formulations of the present invention have a pH of about 5.0 to about 8.0 to provide acceptable stability of the anti-OX40L antibody or its antigen-binding fragment and maintain its biological activity, and are suitable for parenteral administration. In some embodiments, the solution formulations of the present invention have a pH of about 5.0-7.4, such as about 5.0-7.0, about 5.0-6.0, about 5.5-6.0, or about 5.5-5.7. For example, the solution formulations of the present invention have a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.2, about 6.4, about 6.5, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, or a range consisting of any two of these. Preferably, the solution formulation of the present invention has a pH of about 5.0 to about 6.0, for example, about 5.5, about 5.6 or about 5.7.

[0059] The solution formulations of the present invention may optionally contain a buffer to provide the desired pH to the solution formulation. Those skilled in the art will recognize that the choice of buffer is based on the desired pH of the solution formulation and the pKa of the buffer solution, which is within the scope of their expertise.

[0060] In some embodiments, the buffer suitable for use in this invention comprises one or more of the following: acetates, such as sodium acetate, potassium acetate; tartaric acid and / or its salts, such as sodium tartrate, potassium tartrate; carbonates, bicarbonates, such as sodium carbonate, sodium bicarbonate; citric acid and / or its salts, such as sodium citrate, potassium citrate; amino acids and / or their salts, such as amino acids and / or their hydrochloride or sodium salts; phosphates, such as sodium phosphate; hydrogen phosphates, such as sodium dihydrogen phosphate, disodium hydrogen phosphate; Tris (tris(hydroxymethyl)aminomethane); HEPES (4-hydroxyethylpiperazine ethanesulfonic acid); MES buffer; and MOPS buffer. In some embodiments, the buffer comprises one or more of the following: acetates, amino acids or their salts, phosphates, hydrogen phosphates, Tris, HEPES, MES buffer, and MOPS buffer. Preferably, the buffer comprises one or more of the following: acetates, amino acids and / or their salts, phosphates, hydrogen phosphates, and Tris.

[0061] In some embodiments, the buffer comprises or is a salt of an amino acid or its salt, such as an amino acid and / or its hydrochloride or sodium salt, preferably an amino acid and / or its hydrochloride. Preferably, the buffer comprises or is selected from one or more of histidine, histidine hydrochloride, arginine, arginine hydrochloride, glycine, lysine, and monosodium glutamate. More preferably, the buffer comprises or is selected from one or more of histidine, histidine hydrochloride, arginine, and arginine hydrochloride.

[0062] In some embodiments, the buffer comprises either histidine and / or histidine hydrochloride. In some embodiments, the buffer is histidine. In other embodiments, the buffer is histidine hydrochloride. In still other embodiments, the buffer is a combination of histidine and histidine hydrochloride, optionally in a weight ratio of histidine to histidine hydrochloride of about 1:2 to 1:10, preferably about 1:3 to 1:6, more preferably about 1:4 to 1:5, for example about 1:4.42 or about 1:4.46.

[0063] The amount of buffer is the amount that provides the desired pH to the solution formulation, which can be determined by those skilled in the art based on the desired pH of the solution formulation and the pKa of the buffer used. The present invention does not impose any particular limitation on the amount of buffer, as long as it provides the desired pH value or pH range as defined herein. Typically, the concentration of the buffer in the solution formulation is about 1-500 mM, for example about 2-200 mM, about 3-100 mM, about 4-50 mM, about 5-30 mM, about 10-25 mM, for example about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mM or a range consisting of any two of these. When a buffer contains multiple components, the buffer concentration is provided by the combined effect of these components.

[0064] In some embodiments, the concentration of the buffer in the solution formulation is about 0.1-100 mg / mL, for example, about 0.5-60 mg / mL, about 1-30 mg / mL, or about 1-10 mg / mL. In some embodiments, the concentration of the buffer in the solution formulation is a range of about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, or any combination thereof. When the buffer comprises multiple components, the mg / mL concentration is the sum of the mg / mL concentrations of each component.

[0065] In some embodiments, the solution formulation of the present invention comprises about 0.1-10 mg / mL of histidine, for example about 0.5-5 mg / mL, such as a concentration of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 mg / mL or any combination thereof. In some embodiments, the solution formulation of the present invention comprises about 1.55 mg / mL or about 3.10 mg / mL of histidine.

[0066] In some embodiments, the solution formulation of the present invention comprises about 0.1-5 mg / mL histidine and about 0.4-25 mg / mL histidine hydrochloride. Preferably, the concentration of said histidine is about 0.2-4 mg / mL, for example about 0.4-3 mg / mL, about 0.6-2 mg / mL, or about 0.7-0.8 mg / mL, and the concentration of said histidine hydrochloride is about 0.5-20 mg / mL, for example about 1-15 mg / mL, about 2-10 mg / mL, or about 3-4 mg / mL. In some embodiments, the solution formulation of the present invention comprises a combination of about 0.77 mg / mL histidine and about 3.40 mg / mL histidine hydrochloride. In some embodiments, the solution formulation of the present invention comprises a combination of about 0.77 mg / mL histidine and about 3.44 mg / mL histidine hydrochloride.

[0067] In some embodiments, the buffer contained in the solution formulation of the present invention is a combination of histidine and histidine hydrochloride, and the molar concentration of the two in the solution formulation, calculated as histidine, is about 2-200 mM, about 3-100 mM, about 4-50 mM, about 5-30 mM, about 10-25 mM, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mM or any combination thereof. Preferably, the buffer contained in the solution formulation of the present invention is a combination of histidine and histidine hydrochloride, and the molar concentration of both, calculated as histidine, in the solution formulation is about 20, 21, 22, 23, 24, or 25 mM. More preferably, the weight ratio of histidine to histidine hydrochloride having the said molar concentration in the solution formulation is about 1:2 to 1:10, preferably about 1:3 to 1:6, more preferably about 1:4 to 1:5, for example about 1:4.42 or about 1:4.46.

[0068] In some embodiments, the solution formulations of the present invention may optionally contain a surfactant. The surfactants suitable for use in the present invention may be ionic or nonionic surfactants, preferably nonionic surfactants. In some embodiments, the surfactant is a nonionic surfactant, for example selected from: sorbitan fatty acid esters, including polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85) and Span (e.g., Span 20, Span 40, Span 60, Span 65, Span 80, Span 83, Span 85); polypropylene glycol ethylene oxide adducts, including poloxamer (e.g., poloxamer 188); polyoxyethylene alkyl ethers, including benzyl ether (e.g., benzyl ether-30, benzyl ether-35, benzyl ether-52, benzyl ether-56, benzyl ether-58, benzyl ether-76, benzyl ether-28, benzyl ether-92, benzyl ether-96, benzyl ether-98).

[0069] In some embodiments, the surfactant is a polysorbate, such as polysorbate 20 or polysorbate 80.

[0070] Typically, the concentration of surfactants (e.g., polysorbates, such as polysorbate 20 or 80) in solution formulations is about 0.01-10 mg / ml, for example about 0.05-6 mg / ml, about 0.1-3 mg / ml, about 0.2-2 mg / ml, about 0.3-1 mg / ml, about 0.4-0.6 mg / ml, for example about 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml or any combination thereof, preferably about 0.5 mg / ml.

[0071] In some embodiments, the solution formulation of the present invention may optionally include an osmotic pressure regulator. In some embodiments, the osmotic pressure regulator may be selected from one or more of sorbitol, xylitol, mannitol, fructose, glucose, sucrose, mannose, trehalose, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, ammonium sulfate, sodium nitrate, potassium nitrate, phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and calcium phosphate. The amount of osmotic pressure regulator is the amount that makes the osmotic pressure of the solution obtained after reconstitution of the solution formulation or the solid formulation of the present invention close to or equal to the normal osmotic pressure range of an individual, and varies depending on the specific osmotic pressure regulator used. Determining the amount of osmotic pressure regulator is within the scope of skill of those skilled in the art.

[0072] In some embodiments, the osmotic pressure regulator comprises one or more of sorbitol, xylitol, or mannitol, preferably sorbitol. The concentration of the osmotic pressure regulator in the solution formulation can be about 1-200 mg / ml, for example, about 5-150 mg / ml, about 10-100 mg / ml, or about 20-60 mg / ml. In some embodiments, the solution formulation of the present invention comprises about 1-200 mg / ml, for example, about 5-150 mg / ml, about 10-100 mg / ml, or about 20-60 mg / ml of sorbitol. For example, the solution formulation of the present invention comprises sorbitol in the range of about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 mg / ml, or any combination thereof.

[0073] In some embodiments, the osmotic pressure regulator comprises sodium chloride. In some embodiments, the solution formulation of the present invention comprises about 1-100 mg / ml of sodium chloride, for example, about 2-50 mg / ml, about 2-20 mg / ml, about 2-10 mg / ml, or about 3-5 mg / ml of sodium chloride. For example, the solution formulation of the present invention comprises sodium chloride in the range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 50, or 100 mg / ml or any combination thereof. In some embodiments, the solution formulation of the present invention comprises about 4.40 mg / ml of sodium chloride.

[0074] The solution formulation of the present invention may optionally contain a protein stabilizer. In some embodiments, the protein stabilizer may be selected from one or more of the following:

[0075] (1) Sugars and polyols, such as sucrose, lactose, trehalose, maltose, fructose, inulin oligosaccharides, dextran, glucose, maltodextrin, cyclodextrin, dextran, glycerol, mannitol, sorbitol, xylitol, ethylene glycol, etc.

[0076] (2) Amino acids or their salts, such as arginine or its salts, glycine, sodium aspartate, glutamic acid, lysine, etc.

[0077] (3) Complexing agents, such as edetic acid or its salts;

[0078] (4) Surfactants, including ionic or nonionic surfactants, preferably nonionic surfactants;

[0079] (5) Salts, such as sodium chloride, potassium chloride, etc.;

[0080] (6) Polyethylene glycols, such as PEG200, PEG300, PEG400, PEG600, PEG1000, etc.;

[0081] (7) Proteins, such as albumin, casein, mucopolysaccharide proteins, etc.;

[0082] (8) Cyclodextrins, such as hydroxypropyl β-cyclodextrin, etc.

[0083] (9) Other macromolecular compounds, such as heparin, hydroxyethyl starch, polyvinylpyrrolidone, etc.

[0084] In some embodiments, the protein stabilizer may be selected from one or more of the following:

[0085] (1) Sugars and polyols, such as sucrose, lactose, trehalose, maltose, fructose, inulin oligosaccharides, dextran, glucose, maltodextrin, cyclodextrin, dextran, glycerol, mannitol, sorbitol, xylitol, ethylene glycol, etc.

[0086] (2) Amino acids or their salts, such as arginine or its salts, glycine, sodium aspartate, glutamic acid, lysine, etc.

[0087] (3) Complexing agents, such as edetic acid or its salts;

[0088] (4) Surfactants, including ionic or nonionic surfactants, preferably nonionic surfactants;

[0089] (5) Salts, such as sodium chloride, potassium chloride, etc.

[0090] In some embodiments, the protein stabilizer may be selected from one or more of the following: sorbitol, arginine or its hydrochloride, edetate or its salt, polysorbate and sodium chloride.

[0091] Those skilled in the art will understand that some substances of the present invention can perform multiple functions. For example, for the purposes of the present invention, sorbitol and sodium chloride can function as both osmotic pressure regulators and protein stabilizers; some amino acids or their salts can function as both buffers and protein stabilizers.

[0092] In some embodiments, the solution formulation of the present invention may optionally contain a complexing agent, such as edematic acid (EDTA) and / or its salts. In some embodiments, the complexing agent is edematic acid and / or its sodium salt, such as disodium edematic acid or calcium sodium edematic acid. Edematic acid or its salts are used in pharmaceutical preparations as complexing agents, antioxidant synergists, and stabilizers, and their concentration in the solution formulation is typically about 0.005-0.05 mg / mL, for example about 0.005-0.03 mg / mL, for example about 0.01-0.02 mg / mL, for example about 0.01 mg / mL, or about 0.02 mg / mL.

[0093] In some embodiments, the solution formulation of the present invention may optionally contain arginine and / or its salts, such as arginine and / or its hydrochloride. Optionally, the concentration of arginine and / or its salts (such as hydrochloride) in the solution formulation is about 0.1-100 mg / mL, for example about 1-80 mg / mL, about 5-50 mg / mL, or about 15-35 mg / mL. For example, the concentration of said arginine or its salts (such as hydrochloride) in the solution formulation is in the range of about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg / mL or any combination thereof. In some embodiments, the solution formulation of the present invention contains about 15.80, 17.91, or 31.60 mg / mL of arginine or arginine hydrochloride.

[0094] The formulations of the present invention may optionally contain other suitable excipients. In some embodiments, the solution formulations of the present invention may optionally contain solubilizers, co-solvents, antioxidants, adsorption inhibitors, preservatives, local analgesics, etc. As solubilizers and co-solvents, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, etc., may be mentioned. As adsorption inhibitors, human serum albumin, lecithin, polyethylene glycol, etc., may be mentioned. As antioxidants, ascorbic acid, butylated hydroxyanisole, tocopherol, etc., may be mentioned. As preservatives, phenol, cresol, m-cresol, chlorocresol, benzyl alcohol, chlorobutanol, resorcinol, etc., may be mentioned. As local analgesics, benzyl alcohol, chlorobutanol, procaine hydrochloride, etc., may be mentioned. These pharmaceutical excipients and / or additives are known in the art, for example see "The Handbook of Pharmaceutical Excipients", edited by Luo Mingsheng, 2nd edition, 2006; and "The Handbook of Pharmaceutical Excipients, 4th edition, edited by Rowe et al., American Pharmaceutical Association (2003)".

[0095] It should be understood that, unless otherwise specified, the solution formulations of the present invention also include a solvent, including but not limited to water, such as purified water, water for injection, sterile water, double-distilled water, etc. The solution formulations of the present invention can be prepared or formulated using pharmaceutically acceptable solvents or solutions known in the art. The solid formulations of the present invention can be reconstituted using pharmaceutically acceptable solvents or solutions known in the art. Examples of pharmaceutically acceptable solvents or solutions include, but are not limited to, purified water such as ultrapure water, water for injection, sterile water, double-distilled water, physiological saline, Ringer's solution, glucose injection, etc.

[0096] In some embodiments, the solution formulation of the present invention comprises, or is composed of, the following components:

[0097] (a) The anti-OX40L antibody or its antigen-binding fragment;

[0098] (b) A buffer, preferably comprising an amino acid and / or its hydrochloride salt, more preferably comprising histidine and / or histidine hydrochloride;

[0099] (c) Surfactant, preferably nonionic surfactant, more preferably polysorbate;

[0100] (d) Optionally, the osmotic pressure regulator is preferably a polyol (e.g., sorbitol, xylitol or mannitol) or sodium chloride;

[0101] (e) Optionally, a complexing agent, such as edetate and / or its salts (e.g., sodium salt);

[0102] (f) Optionally, protein stabilizers, such as arginine and / or its hydrochloride;

[0103] (g) Solvent, preferably water;

[0104] The solution formulation has a pH of about 5.0-8.0, preferably about 5.0-7.4, such as about 5.0-7.0, about 5.0-6.0, about 5.5-6.0, or about 5.5-5.7, such as about 5.5, about 5.6, or about 5.7. Optionally, the type and / or amount of the component are as described herein.

[0105] In some embodiments, the solution formulation of the present invention comprises, or is composed of, the following components:

[0106] (a) The anti-OX40L antibody or its antigen-binding fragment, optionally at a concentration of about 1-500 mg / mL;

[0107] (b) Histidine and / or histidine hydrochloride, optionally at a concentration of about 0.1-100 mg / mL, for example about 0.5-60 mg / mL, about 1-30 mg / mL, or about 1-10 mg / mL; or at a concentration of about 1-500 mM, for example about 2-200 mM, about 3-100 mM, about 4-50 mM, about 5-30 mM, or about 10-25 mM, wherein the weight ratio of said histidine to histidine hydrochloride is optionally about 1:2 to 1:10, preferably about 1:3 to 1:6, more preferably about 1:4 to 1:5, for example about 1:4.42 or about 1:4.46;

[0108] (c) Polysorbate, optionally at a concentration of about 0.01-10 mg / ml, for example about 0.05-6 mg / ml, about 0.1-3 mg / ml, about 0.2-2 mg / ml, about 0.3-1 mg / ml, about 0.4-0.6 mg / ml;

[0109] (d) Optionally, a polyol (e.g., sorbitol, xylitol or mannitol), preferably sorbitol, is used at a concentration of about 1-200 mg / ml, for example, about 5-150 mg / ml, about 10-100 mg / ml or about 20-60 mg / ml;

[0110] (e) Optionally, edetate and / or its sodium salt, such as disodium edetate or calcium sodium edetate, at a concentration of about 0.005-0.05 mg / ml, such as about 0.005-0.03 mg / ml, such as about 0.01-0.02 mg / ml;

[0111] (f) Optionally, sodium chloride, optionally at a concentration of about 1-100 mg / ml, for example about 2-50 mg / ml, 2-20 mg / ml, 2-10 mg / ml, or 3-5 mg / ml;

[0112] (g) Optionally, arginine and / or its hydrochloride, optionally at a concentration of about 0.1-100 mg / mL, for example about 1-80 mg / mL, about 5-50 mg / mL, about 15-35 mg / mL;

[0113] (h) Solvent, preferably water;

[0114] The solution formulation has a pH of about 5.0-6.0, for example, about 5.5, about 5.6 or about 5.7.

[0115] In some embodiments, the solution formulation of the present invention comprises, or is composed of, the following components:

[0116] (a) Approximately 100-150 mg / mL of anti-OX40L antibody or its antigen-binding fragment;

[0117] (b) about 0.5-5 mg / mL of histidine, or a combination of about 0.7-0.8 mg / mL of histidine and about 3-4 mg / mL of histidine hydrochloride; or about 5-30 mM or about 10-25 mM of histidine and / or histidine hydrochloride, based on the concentration of histidine, optionally in a weight ratio of about 1:2 to 1:10, preferably about 1:3 to 1:6, more preferably about 1:4 to 1:5, for example about 1:4.42 or about 1:4.46;

[0118] (c) Polysorbate 80, optionally at a concentration of about 0.4-0.6 mg / ml;

[0119] (d) Polyols (e.g., sorbitol, xylitol or mannitol), preferably sorbitol, optionally at a concentration of about 20-60 mg / ml;

[0120] (e) Edesmonic acid and / or its sodium salt, such as disodium edetate or calcium sodium edetate, optionally at a concentration of about 0.01-0.02 mg / ml;

[0121] (f) Optionally, sodium chloride, optionally at a concentration of about 3-5 mg / ml;

[0122] (g) Optionally, arginine and / or its hydrochloride, optionally at a concentration of about 15-35 mg / ml;

[0123] (h) Solvent, preferably water;

[0124] The solution formulation has a pH of about 5.0-6.0, for example, about 5.5, about 5.6 or about 5.7.

[0125] In some embodiments, the solution formulation of the present invention comprises, or is composed of, the following components:

[0126] (a) Approximately 100 mg / mL of anti-OX40L antibody or its antigen-binding fragment;

[0127] (b) about 0.77 mg / ml of histidine and about 3.40 mg / ml of histidine hydrochloride; or about 0.77 mg / ml of histidine and about 3.44 mg / ml of histidine hydrochloride; or a combination of histidine and histidine hydrochloride, wherein the concentration of histidine in the solution is about 20 mM, 21 mM or 22 mM, optionally the weight ratio of said histidine to histidine hydrochloride is about 1:4.42 or about 1:4.46;

[0128] (c) Approximately 0.5 mg / mL of polysorbate 80;

[0129] (d) Approximately 50 mg / mL of sorbitol;

[0130] (e) Sodium edetate at approximately 0.02 mg / ml;

[0131] (f) Solvent, preferably water;

[0132] The solution preparation has a pH of about 5.0-8.0, preferably about 5.0-6.0, for example about 5.5, 5.6 or 5.7.

[0133] In some embodiments, the solution formulation of the present invention comprises, or is composed of, the following components:

[0134] (a) Approximately 150 mg / mL of anti-OX40L antibody or its antigen-binding fragment;

[0135] (b) about 0.77 mg / ml of histidine and about 3.40 mg / ml of histidine hydrochloride; or about 0.77 mg / ml of histidine and about 3.44 mg / ml of histidine hydrochloride; or a combination of histidine and histidine hydrochloride, wherein the concentration of histidine in the solution is about 20 mM, 21 mM or 22 mM, optionally the weight ratio of said histidine to histidine hydrochloride is about 1:4.42 or about 1:4.46;

[0136] (c) Approximately 40 mg / mL of sorbitol;

[0137] (d) Sodium edetate at approximately 0.02 mg / ml;

[0138] (e) Approximately 0.5 mg / mL of polysorbate 80;

[0139] (f) Solvent, preferably water;

[0140] The solution preparation has a pH of about 5.0-8.0, preferably about 5.0-6.0, for example about 5.5, 5.6 or 5.7.

[0141] In some embodiments, the solution formulations of the present invention are those disclosed in the examples. Furthermore, embodiments based on the formulations disclosed in the examples, but with variations in the concentration values ​​of the components in these formulations by 50%, 40%, 30%, 20%, 10%, or 5%, and in the pH value by 5%, 4%, 3%, 2%, 1%, or 0.5%, are also included in this application.

[0142] The solution formulations of the present invention are typically administered via parenteral routes, such as injection (e.g., intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, peritoneal injection, spinal injection, etc.), transdermal, transmucosal, nasal, or pulmonary routes. Preferably, the solution formulations of the present invention are injectable solution formulations, and are preferably administered via intravenous injection, such as intravenous infusion.

[0143] The solution formulations of the present invention can be loaded into pre-filled syringes (including pre-filled injection pens) for use. Therefore, in another aspect, pre-filled syringes containing a solution formulation as defined herein are provided. In some embodiments, the pre-filled syringe is used for intravenous or subcutaneous injection, such as a subcutaneous bolus.

[0144] The solution formulations of the present invention can also be prepared into solid dosage forms, such as freeze-dried or spray-dried formulations, by a solidification process (e.g., freeze-drying or spray-drying). Therefore, in another aspect, any other form of pharmaceutical dosage form, such as a solid dosage form, for example a freeze-dried or spray-dried formulation, obtained from the solution formulations of the present invention is provided.

[0145] In some embodiments, the solid dosage form is a lyophilized powder for injection. The solid dosage form can be reconstituted into a solution preparation with a suitable solvent (e.g., water, such as purified water like ultrapure water or water for injection) before use. This reconstituted preparation also belongs to the solution preparations of this invention.

[0146] When referring to formulations of the present invention, it includes the solution formulations of the present invention or any other forms obtained therefrom, such as pre-filled syringes or pre-filled injection pens or solid formulations, such as spray-dried formulations or freeze-dried formulations.

[0147] The formulation of the present invention is stable. Therefore, in this document, the formulation is a stable formulation.

[0148] The solution formulation of the present invention is stable. Therefore, in this document, the solution formulation is a stable solution formulation.

[0149] The formulations of the present invention (e.g., the solution formulations of the present invention or any other form of pharmaceutical formulation obtained therefrom, such as solid formulations) can be stored stably for extended periods, such as at least 24 months or longer. In some embodiments, the formulations of the present invention can be stored at temperatures from about -80°C to about 45°C, for example, -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 38°C, about 40°C, about 42°C, or about 45°C, for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, and are stable.

[0150] In some embodiments, after storage, the stability of the solution formulation of the present invention is visually inspected, and the solution formulation of the present invention remains clear to slightly opalescent in appearance, is a colorless to pale yellow liquid, and is free of foreign matter. In some embodiments, visual inspection under a clarity meter shows that no visible foreign matter is present in the formulation.

[0151] In some embodiments, the stability of the solution formulation of the present invention is checked by measuring changes in protein content after storage, wherein, for example by ultraviolet spectrophotometry (UV), the rate of change in protein content relative to the initial value on day 0 of storage is not more than 10%, for example 7-8%, more preferably not more than 5%.

[0152] In some embodiments, the stability of the solution formulation of the present invention is checked by measuring the change in purity of the solution formulation after storage, wherein the change in monomer purity relative to the initial value on day 0 of storage by size exclusion high performance liquid chromatography (SEC-HPLC) is not more than 10%, for example not more than 5%, 4%, 3%, for example not more than 1-2%, preferably not more than 1%.

[0153] In some embodiments, the stability of the solution formulation of the present invention is checked after storage by measuring the change in purity of the solution formulation of the present invention, wherein the change in monomer purity by non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS) does not exceed 10%, for example, not more than 5%, 4%, 3% or 2%.

[0154] In some embodiments, the stability of the solution formulation of the present invention is detected by imaging capillary isoelectric focusing electrophoresis (iCIEF) after storage, wherein the sum of the changes in the charge variants (main component, acidic component and basic component) of the antibody relative to the initial value on day 0 of storage does not exceed 30%, for example, not more than 20%, not more than 10%, not more than 5%, not more than 4% or not more than 3%.

[0155] In some embodiments, the binding activity of the solution formulation of the present invention to the antigen is detected by ELISA after storage, wherein the biological activity is in the range of 50-150% of the initial value relative to the initial value on day 0 of storage.

[0156] The anti-OX40L antibody or its antigen-binding fragment of the present invention can be used to prevent or treat diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway.

[0157] Therefore, in another aspect, the present invention provides formulations, such as the solution formulations of the present invention or solid formulations obtained therefrom, for the prevention or treatment of diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway.

[0158] In another aspect, the use of the formulations of the present invention, such as the solution formulations of the present invention or the solid formulations obtained therefrom, is provided for the prevention or treatment of diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway.

[0159] In another aspect, the use of the formulations of the present invention, such as the solution formulations of the present invention or the solid formulations obtained therefrom, is provided in the preparation of medicaments for the prevention or treatment of diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway.

[0160] On the other hand, methods are provided for preventing or treating diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway in an individual, the methods comprising administering to the individual an effective amount of a formulation of the present invention, such as a solution formulation of the present invention or a solid formulation obtained therefrom, optionally in combination with one or more other therapies, such as pharmaceutical therapy and / or surgical treatment. In some embodiments, the methods comprise administering to the individual by injection, such as intravenous or subcutaneous injection, of the formulation of the present invention.

[0161] On the other hand, a kit is provided comprising the formulation of the present invention, such as the solution formulation of the present invention or the solid formulation obtained therefrom, and instructions for use of said formulation for the prevention or treatment of diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway. Optionally, when said formulation is a solid formulation, the kit further comprises a reconstituted solvent, preferably water, such as water for injection, in a separate container.

[0162] In another aspect, the formulations of the present invention, such as the solution formulations of the present invention or the solid formulations obtained therefrom, are provided in combination with one or more other therapies, such as pharmaceutical treatment and / or surgical treatment, for the uses described herein, such as for the prevention or treatment of diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway, or for the methods described herein.

[0163] In another aspect, a method for preparing the solution formulation of the present invention is provided.

[0164] In all the foregoing aspects, “diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway” as used herein refers to any disease associated with abnormal activation of the OX40 / OX40L signaling pathway. Therefore, the formulations of the present invention can reduce the associated immune response by blocking OX40L-OX40 signaling, thereby inhibiting T cell activation and reducing diseases or symptoms associated with autoimmune overreaction. In some embodiments, patients with said disease or condition have abnormal activation of the OX40 / OX40L signaling pathway compared to healthy individuals. In some embodiments, patients with said disease or condition have OX40L / OX40-mediated immune system overreaction compared to healthy individuals. In some embodiments, patients with said disease or condition have increased OX40L expression on cells (e.g., antigen-presenting cells) and / or increased OX40 expression on cells (e.g., T cells, such as activated T cells) in patients with said disease or condition, for example, compared to corresponding cells in healthy individuals. In some embodiments, said disease or condition is selected from autoimmune diseases or graft-versus-host disease. In some implementation plans, autoimmune diseases include, but are not limited to, allergic dermatitis, asthma, systemic lupus erythematosus, Schogren's syndrome, immune thrombocytopenic purpura, multiple sclerosis, lupus nephritis, amyotrophic lateral sclerosis, rheumatoid arthritis, non-rheumatoid arthritis, contact dermatitis, hyperIgE syndrome, inflammatory bowel disease, myasthenia gravis, Greaves' disease, hemolytic anemia, psoriasis, atopic dermatitis, allergic asthma, or idiopathic inflammatory disease (CN106459196B).

[0165] It is understood that the solution formulations described herein, along with their various embodiments, features, and advantages, can be equally applied to other aspects of the invention, including aspects not directly described, and their various embodiments, features, and advantages.

[0166] definition

[0167] It should be understood that the terminology used herein is for the purpose of describing embodiments of the invention only and is not intended to limit the scope of the invention. The terminology used in this application has the meanings defined below, unless otherwise stated or the context clearly contradicts it. Terms not explicitly defined in this application have the meanings commonly understood by those skilled in the art. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0168] The terms “a,” “an,” “the,” and similar terms used in this application shall be understood to include both the singular and the plural, unless otherwise stated or the context clearly contradicts it.

[0169] The term "about" when used in conjunction with a numerical value means to cover a range of values ​​having a lower limit that is 5%, 4%, 3%, 2%, or 1% smaller than the specified numerical value and an upper limit that is 5%, 4%, 3%, 2%, or 1% larger than the specified numerical value. When the term "about" is used in conjunction with a numerical range, such as in "about 5.0-7.0," it is intended to cover a numerical range whose lower limit is 5%, 4%, 3%, 2%, or 1% smaller than the lower limit of the numerical range associated with "about," and whose upper limit is 5%, 4%, 3%, 2%, or 1% larger than the upper limit of the numerical range associated with "about." When the term "about" is used in conjunction with multiple numerical values ​​or numerical ranges, it indicates that each of the stated numerical values ​​or numerical ranges is used in conjunction with the term "about." For example, the expression "about 5.5, 5.6, or 5.7" is intended to cover the cases of "about 5.5," "about 5.6," and "about 5.7."

[0170] As used herein, the term “and / or” means any one of the options or two or more or all of the options.

[0171] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting essentially of the stated elements, integers, or steps, and situations consisting solely of the stated elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0172] The term "consistently composed of..." 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. The term "w / w" means by weight.

[0173] As used herein, “OX40L” refers to any naturally occurring OX40L polypeptide (e.g., human OX40L polypeptide) or a variant thereof. The term “OX40L” encompasses “full-length” untreated OX40L polypeptides as well as any form of OX40L polypeptide produced by intracellular treatment. The term also encompasses naturally occurring variants of OX40L, such as those encoded by splice variants and allelic variants. The OX40L polypeptides described herein can be isolated from a variety of sources, such as human tissue or from another source, such as cynomolgus monkeys, or prepared by recombinant or synthetic methods. In one embodiment of the invention, the human OX40L protein is shown as (CD252, Uniport: P23510). In one embodiment, the human OX40L gene is shown as (Uniport: P29279). In one embodiment of the invention, the cynomolgus monkey OX40L protein is shown as (uniport: A0A1D5QQH7). In one embodiment of the invention, the human OX40L protein has the amino acid sequence shown in SEQ ID NO: 14.

[0174] General information on the amino acid and nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0175] The terms "full-length antibody" or "complete antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that has only heavy chains and lacks light chains, a full-length antibody consists of two heavy chains (H) linked together by disulfide bonds. For a conventional four-chain IgG antibody, the heavy chain of a full-length antibody typically consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The light chain of a full-length antibody consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region 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 term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0176] The term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It contains 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, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), and nanobodies. 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. Fab' monomers are essentially Fab fragments with hinge regions. Fv fragments consist of the VL and VH domains of the antibody's single arm. The two domains VL and VH of the Fv fragment can be encoded by independent genes, but they can also be produced as a single protein chain by using a recombinant approach, connecting the two domains with a synthetic linker peptide, and pairing the VL and VH regions in the single protein chain to form a single-chain Fv (scFv).

[0177] The complementarity-determining region (CDR) or CDR is a region in the antibody variable region 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 region are called HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable region are called 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) (www.imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers 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).

[0178] In some embodiments, the CDRs in the heavy chain variable region and the light chain variable region of the antibody of the present invention are determined according to Kabat, Chothia, AbM or IMGT or any combination thereof (e.g., a combination of Kabat and Chothia). For example, HCDR1 of the heavy chain variable region is determined according to the AbM scheme, and HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region are determined according to the Kabat scheme.

[0179] An example CDR definition scheme is as follows:

[0180] The following is a calculation of sequence identity between sequences.

[0181] The "percentage of identity (%)" for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification, after comparing the candidate sequence with the specific amino acid sequence shown herein and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a considerable degree of identity with respect to the antibody molecules and their sequences specifically disclosed herein, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. These variants may contain conserved changes.

[0182] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human 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 (i.e., replacing the non-binding portion of the variable region with the corresponding portion of the human antibody).

[0183] "Separated" antibodies refer to antibodies that have been separated from components of their natural environment. In some embodiments, antibodies are purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0184] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0185] The term "pharmaceutical excipients" refers to pharmaceutically acceptable carriers, diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, buffers, or stabilizers that are applied together with the active substance.

[0186] The term "lyophilized formulation" or "freeze-dried preparation" refers to a composition obtained or obtainable by freeze-drying a solution preparation, sometimes also referred to as a lyophilized powder. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0187] The term "kit" refers to a kit containing one or more individual pharmaceutical compositions or formulations, wherein at least one pharmaceutical composition or formulation contains an effective amount of an anti-OX40 antibody or its antigen-binding fragment, and preferably instructions for use for the prevention or treatment of diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway. The kit may also contain other therapeutic agents with the same or different activities. It is understood that when a kit contains multiple individual pharmaceutical compositions or formulations, each of these compositions or formulations may contain different dosages and / or be administered via different routes.

[0188] The term "injection" means 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 subcutaneous injection, particularly subcutaneous bolus injection. When used for intravenous infusion, the formulations of the present invention may be reconstituted or diluted with "reconstituted solvents" such as 0.9% sodium chloride injection and / or 5% glucose injection, if desired.

[0189] The term "effective amount" refers to the amount of antibody or antigen-binding fragment thereof that produces the desired effect in an individual after administration of the formulation of the present invention. Depending on the purpose of use, the "effective amount" can be a preventive effective amount or a therapeutic effective amount. A "therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. A therapeutic effective amount is also an amount in which any toxic or harmful effects of the formulation of the present invention are less than the beneficial therapeutic effects. A "preventive effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired preventive outcome. Generally, because the preventive dose is used in the subject before or at an early stage of the disease, the preventive effective amount will be less than the therapeutic effective amount.

[0190] 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.

[0191] The term "treatment" refers to the reduction, cessation, relief, or reversal of an existing disease or its symptoms, or the slowing of the progression of an existing disease or its symptoms, including palliative and curative treatment.

[0192] 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.

[0193] The term "buffer" refers to a substance that, when a certain amount of acid or base is added, prevents a significant change in the pH of a solution, thereby maintaining the pH value within a desired range.

[0194] 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.

[0195] The term "pharmaceutically acceptable" means that there are no unacceptable toxicities, irritants, or side effects for animals, especially humans.

[0196] The term "drug combination" refers to a non-fixed or fixed combination, including but not limited to a pillbox. The term "non-fixed combination" means that active ingredients (e.g., (i) the formulations of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. In some embodiments, the antibodies of the present invention used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in separate formulations, which may be the same or different.

[0197] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0198] Preparation method

[0199] On the other hand, a method for preparing a solution formulation of the anti-OX40L antibody or its antigen-binding fragment of the present invention is also provided. Specifically, the solution formulation of the anti-OX40L antibody or its antigen-binding fragment of the present invention is prepared by a method comprising the following steps:

[0200] (a) Dissolve the buffer in water to obtain a buffer solution having a desired pH;

[0201] (b) Add the anti-OX40L antibody or its antigen-binding fragment to the buffer solution of step (a);

[0202] (c) Add a surfactant to the solution from step (b) to obtain the solution formulation.

[0203] When the formulation of the present invention also contains other excipients (e.g., osmotic pressure regulators, complexing agents, etc.), these excipients are added to the buffer solution in step (a).

[0204] In some embodiments, the components used in the method of the present invention and their amounts are as defined in the solution formulation of the present invention, and will not be repeated here.

[0205] In some embodiments, the anti-OX40L antibody or its antigen-binding fragment is isolated and purified, and optionally added to an ultrafiltration centrifuge tube for centrifugation concentration.

[0206] In some embodiments, the anti-OX40L antibody or its antigen-binding fragment is added to the buffer solution of step (a) by ultrafiltration replacement.

[0207] In some implementations, in step (b), the protein concentration is adjusted to the desired concentration value after the replacement is completed.

[0208] In some embodiments, the pH of the solution may optionally be detected after each step of the method of the present invention, and the pH may be adjusted to a desired range if necessary. For example, in step (b), after adding the anti-OX40L antibody or its antigen-binding fragment to the buffer solution of step (a), the pH may optionally be adjusted to a desired range. Also, for example, in step (c), after adding a surfactant to the solution, the pH may optionally be adjusted to a desired range.

[0209] In some embodiments, the method of the present invention further includes:

[0210] (d) Optionally, the solution from step (c) is aseptically filtered; and

[0211] (e) Optionally, the solution from step (d) is dispensed into vials (e.g., ampoules or vials) and sealed to obtain the finished product.

[0212] In some implementations, in step (e), the solution is dispensed into vials, capped with rubber stoppers and aluminum-plastic caps, to obtain the finished product.

[0213] On the other hand, a method for preparing a lyophilized formulation is also provided, comprising the following steps: lyophilizing the solution formulation obtained in step (c) above to obtain a lyophilized formulation. Optionally, additional excipients, such as a lyophilization protectant, may be added to the solution formulation obtained in step (c) before lyophilization. Suitable lyophilization protectants are well known in the art.

[0214] On the other hand, a method for preparing a spray-dried formulation is also provided, comprising the following steps: spray-drying the solution formulation obtained in step (c) above to obtain a spray-dried formulation. Spray drying methods are known in the art.

[0215] It is understood that the order of addition of each component in the preparation method of the present invention 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. Attached Figure Description

[0216] Figure 1 shows the affinity of the anti-OX40L antibody, its blocking activity against OX40-OX40L, and its effect on the activity of Jurkat-NFκB luciferase overexpressing OX40.

[0217] Figure 2 shows the effect of anti-OX40L antibody on T cell activation;

[0218] Figure 3 shows the changes in IL5 and IL13 secretion after treatment with anti-OX40L antibody during DC-induced Th2 differentiation;

[0219] Figure 4 shows the changes in body weight and survival curves of mice in the GvHD mouse model;

[0220] Figure 5 shows the proportion of CD4-positive T cells in the blood of mice in the GvHD mouse model on day 14.

[0221] Figure 6 shows the results of the pre-formulation study, where: A: Purity (SEC-HPLC method) trend; B: Purity (non-reducing CE-SDS method) trend; C: Charge variant-acidic component (iCIEF method) trend; D: Charge variant-principal component (iCIEF method) trend.

[0222] Figure 7 shows the curves of the prescription screening study results, where: A: Purity (SEC-HPLC method) trend graph; B: Purity (non-reducing CE-SDS method) trend graph; C: Charge variant-acidic component (iCIEF method) trend graph; D: Charge variant-principal component (iCIEF method) trend graph.

[0223] Figure 8 shows the trend curves of the prescription determination results, where: A: Purity (SEC-HPLC method) trend curve; B: Purity (non-reducing CE-SDS method) trend curve; C: Charge variant-acidic component (iCIEF method) trend curve; D: Charge variant-principal component (iCIEF method) trend curve. Example

[0224] The following embodiments are provided to further illustrate the present invention. It should be understood that these embodiments are merely for the purpose of better understanding the invention and are not intended to limit the scope of the invention in any way. All changes or equivalent substitutions that do not depart from the inventive concept are included within the scope of the invention.

[0225] Unless otherwise specified, all materials and reagents used are commercially available or prepared according to methods known in the art. The raw materials used in the examples are commercially available and can be used directly without further purification, unless otherwise stated.

[0226] Example 1. Preparation and property verification of HZ22 antibody

[0227] Equipment: Thermo Fisher Multiskan-FC (Examples 1.1-1.4)

[0228] Equipment: SPARK real-time microplate reader (Examples 1.1-1.2)

[0229] Equipment: BD FACSymphony A3 Flow Cytometer (Example 1.5)

[0230] Reagents and raw materials:

[0231] 1.1 Preparation of anti-OX40L antibody molecules

[0232] The sequence of the anti-OX40L antibody HZ22 is available in the sequence information, which is from PCT / CN2024 / 109523.

[0233] The light and heavy chain variable region gene fragments of the anti-OX40L antibody HZ22 were ligated into the pcDNA3.1 vector using homologous recombinase, wherein the constant region was selected as the IgG4 isotype (heavy chain constant region: containing the S228P mutation, SEQ ID NO:11; light chain constant region: SEQ ID NO:12), and expression plasmids of light chain and heavy chain antibodies were obtained.

[0234] Pass HEK293 cells (Invitrogen) to the required transfection volume, adjusting the cell density to 1.5 × 10⁻⁶ cells the day before transfection. 6 Cells / ml. The cell density on the day of transfection was approximately 3 × 10⁻⁶ cells / ml. 6 Cells / ml. Take 1 / 10 (v / v) of Opti-MEM medium (Gibco catalog number: 31985-070) as transfection buffer, add the expression plasmid pcDNA3.1 containing the heavy and light chains of the humanized antibody (which contains nucleic acids encoding the heavy and light chains of the HZ22 antibody), mix well, and filter through a 0.22 μm filter. Add appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid from the previous step (plasmid to PEI mass ratio of 1:3), mix well, and incubate at room temperature for 10 min to obtain the DNA / PEI mixture. Gently pour the DNA / PEI mixture into HEK293 cells and mix well. Incubate at 37°C and 8% CO2 for 24 h. Then, add VPA (Sigma, catalog number: P4543-100G) to a final concentration of 2 mM and 2% (v / v) feed solution (1 g / L Phytone Peptone + 1 g / L Difco Select Phytone) and continue culturing for 6 days.

[0235] After cell culture, the cell culture medium was centrifuged at 13000 rpm for 20 min, and the supernatant was collected. Following the manufacturer's instructions, the supernatant was purified using a pre-packed Hitrap Mabselect Sure (GE, 11-0034-95) column, and the concentration was determined. 100 μg of the purified protein was taken, and the concentration was adjusted to 1 mg / mL. The protein purity was determined using a gel filtration chromatography column SW3000 (TOSOH, catalog number: 18675). The results showed that a high-purity chimeric antibody was obtained.

[0236] 1. HZ22 antibody affinity assay

[0237] The affinity of the HZ22 antibody for the antigen (human and cynomolgus monkey OX40L) was determined using the ForteBio assay and expressed as the equilibrium dissociation constant (KD). The results are shown in Table 1.

[0238] Table 1. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0239] 1.2 HZ22 antibody blocks OX40 / OX40L protein binding and blocks OX40 signaling activation.

[0240] The inhibitory effects of HZ22 antibody on OX40L at both the molecular and cellular levels were tested. In a co-culture system of CHO-OX40L overexpressing cells and Jurkat-OX40-NFκB-Luc reporter cells, the addition of HZ22 antibody blocked the binding of OX40L on the surface of CHO cells and OX40 on the surface of Jurkat-OX40-NFκB-Luc reporter cells, thereby inhibiting the activation of downstream NFκB and luciferase production in Jurkat cells and reducing the fluorescence signal of the system.

[0241] CHO-OX40L cell preparation

[0242] The encoding nucleic acid sequence of Human OX40L (SEQ ID NO:14) was constructed in the pXC17.4 plasmid and digested with PvuI (NEB) to obtain a linear vector. Healthy GS-CHO cells were electroporated with 40 μg of the vector and 1 × 10^7 cells. After electroporation, the cells were transferred to fresh GS-CHO culture medium without GlutaMax. Once the GS-CHO cells were growing normally, L-methionine sulfoxide was added to the culture medium for cell selection. After the cell viability recovered to over 95%, flow cytometry was used to sort the cells, yielding GS-CHO-OX40L (CHO-OX40L).

[0243] ELISA detection of antibody blocking OX40 / OX40L protein binding levels and

[0244] Experimental Procedure: Dilute OX40L recombinant protein to 1 μg / ml using citrate-buffered saline. Add 100 μl to each well of a 96-well microplate and incubate overnight at 4°C. The next day, remove the coated microplate, wash three times with PBST buffer (1×PBS, 0.05% Tween), blot dry, and add blocking buffer (1×PBS, 0.05% Tween, 1% BSA) to the 96-well microplate. Incubate at room temperature for 2 hours. Wash three times with PBST buffer and blot dry. Dilute OX40-Avi to 1 μg / ml using blocking buffer, and simultaneously dilute the antibody to be tested using blocking buffer. Add 50 μl of diluted OX40-Avi and 50 μl of diluted antibody to the 96-well microplate, respectively, and incubate at room temperature for 2 hours. Wash three times with PBST buffer and blot dry. Dilute Streptavidin-HRP (1:3000) with blocking buffer and add 100 μl to each well of a 96-well microplate. Incubate at room temperature in the dark for 1 h. Wash three times with PBST buffer and blot dry. Add 100 μl of TMB chromogenic solution to each well for development, and stop development with stop solution. Read the OD450 absorbance using a microplate reader.

[0245] Reporter gene system detection of antibody blocking OX40 signaling activation

[0246] Experimental Procedure: Prepare well-grown OX40L overexpressing cells (CHO-OX40L) and OX40 overexpressing reporter cells (Jurkat-OX40-NFκB-Luc). Count the cells. Adjust the CHO-OX40L cell density to 5.5 × 10^4 / ml using 1640 complete culture medium, and dilute the Jurkat-OX40-NFκB-Luc cell density to 2.2 × 10^6 / ml. Simultaneously, dilute the antibody using 1640 complete culture medium. Add 45 μl of CHO-OX40L and 45 μl of Jurkat-OX40-NFκB-Luc cells to each well of a 96-well white plate, along with 10 μl of antibody. Incubate the 96-well white plate in a 37°C cell culture incubator for 6 h. After incubation, add 100 μl of Bio-Glo Reagent to each well, incubate at room temperature in the dark for 10 min, and then read the fluorescence value using a multi-mode microplate reader.

[0247] Figure 1 shows the experimental results. The antibody KY1005 is the antibody disclosed in US10654935B2.

[0248] 1.3 Inhibition of primary T cell activation by anti-OX40L antibody

[0249] In a co-culture system of CHO-OX40L overexpressing cells and primary T cells, the effect of anti-OX40L antibody on T cell activation was examined, and the inhibitory effect was reflected by measuring the secretion level of IL-2 in the culture system.

[0250] The experimental procedure is as follows: Dilute OKT3 to 1 μg / ml, add 100 μl to each well of a 96-well U-plate, and incubate overnight at 4°C. The next day, remove the liquid from the 96-well U-plate, wash the 96-well U-plate three times with 1×PBS, and blot dry. Use the Pan-T cell isolation kit (Miltenyi Biotec) to isolate Pan T cells from PBMCs according to the manufacturer's instructions, and adjust the cell density to 1×10⁶ cells / well using RPMI medium 1640 complete culture medium (RPMI medium 1640 + 5% FBS). 6 = / ml, and add anti-human CD28. Dilute CHO-OX40L cells to 0.8×10⁵ / ml with 1640 complete culture medium, and dilute the antibody to be tested with 1640 complete culture medium. Add 100μl Pan T cells, 50μl CHO-OX40L and 50μl antibody to be tested to a patted-dry 96-well U plate. Incubate the 96-well U plate in a 37°C cell culture incubator for 3 days. After 3 days, remove the 96-well U plate, centrifuge at 500×g for 10 min, and collect the culture supernatant. Use IL-2 ELISA (Human IL-2 DuoSet ELISA, R&D) to detect the IL-2 content in the supernatant to indicate the activation status of T cells.

[0251] Figure 2 shows the experimental results. As can be seen from Figure 2, the blocking antibody against OX40L can significantly inhibit the activation level of T cells in vitro.

[0252] 1.4 Effect of anti-OX40L antibody on DCs-induced T cell differentiation into Th2 cells

[0253] Existing research has shown that thymic stromal lymphopoietin (TSLP) can induce the expression of OX40L on dendritic cells, thereby promoting the differentiation of T cells into Th2 cells during antigen presentation and secreting Th2 cell-related factors IL-4, IL-5 and IL-13.

[0254] In this experiment, primary dendritic cells (DCs) isolated from the culture were stimulated with recombinant hTSLP (Sino Biological), and then the DCs were combined with the isolated DCs. CD4 T cells were co-cultured for 6 days, and then anti-CD3 / anti-CD28 antibodies were added to further activate the T cells. Anti-OX40L antibody or isotype-control was added to the entire culture system, and the inhibitory effect of the antibody was evaluated by detecting the secretion levels of IL-5 and IL-13.

[0255] Experimental steps:

[0256] mDCs were isolated using the EasySep Human Myeloid DC Enrichment Kit (Stemcell).

[0257] Remove PBMCs, thaw rapidly, and wash twice with 1×PBS; resuspend PBMCs in MACS Buffer to 5×10⁷ cells / ml; add 15 μl / ml FcR blocker, 50 μl / ml Myeloid DC Enrichment Cocktail Component A, and 50 μl / ml DC Enrichment Cocktail Component B to the system, mix well, and incubate at room temperature in the dark for 30 min; remove the separated magnetic beads, vortex to mix, and add to the system at 100 μl / ml, mix well, and incubate at room temperature in the dark for 10 min; add MACS Buffer to the system to a volume of 2.5 ml, and transfer to a flow cytometry tube; place the flow cytometry tube on a magnetic rack and wait at room temperature for 5 min; transfer the liquid to a new flow cytometry tube, place the new flow cytometry tube in the magnetic field, and wait at room temperature for 5 min; transfer the liquid to a 15 ml tube, centrifuge at 500×g for 10 min; resuspend mDCs in X-VIVO 15, centrifuge at 500×g for 10 min. mDCs were resuspended in X-VIVO 15 (Lonza) and the cell density was adjusted to 1×10^6 cells / ml. Recombinant hTSLP was added to the mDC suspension to a final concentration of 20 ng / ml, and the cells were seeded into 200 μl per well of an ultra-low adsorption 96-well U-plate. The mDC cells in the ultra-low adsorption 96-well U-plate were incubated at 37°C for 2 days.

[0258] Remove mDC cells from the incubator and centrifuge at 500×g for 10 min; remove the supernatant, resuspend the cells in fresh X-VIVO 15 medium, and centrifuge at 500×g for 10 min; resuspend the cells in fresh X-VIVO 15 medium again, centrifuge at 500×g for 10 min, and remove the supernatant; adjust the mDC concentration to 160,000 cells / ml using X-VIVO 15.

[0259] Naive CD4+ T cells were isolated using the EasySep Human Naive CD4+ T Cell Isolation Kit (Stemcell): PBMCs were removed, rapidly thawed, and washed twice with 1×PBS; PBMCs were resuspended in MACS Buffer to 5×10⁷ cells / ml; 50 μl / ml of Biotinylated Anti-CD45RO Antibody and 50 μl / ml of Isolation Cocktail were added, mixed, and incubated at room temperature for 5 min; the separated magnetic beads were removed, vortexed, and added to the system at 50 μl / ml, mixed, and incubated at room temperature for 5 min; the volume of the system was increased to 2.5 ml by adding MACS Buffer and transferred to a flow cytometry tube; the flow cytometry tube was placed on a magnetic rack and incubated at room temperature for 5 min; the liquid was transferred to a new flow cytometry tube, and the new flow cytometry tube was placed in a magnetic rack and incubated at room temperature for 5 min; the liquid was transferred to a 15 ml tube and centrifuged at 500×g for 10 min; the T cells were resuspended in X-VIVO 15 and centrifuged at 500×g for 10 min. Naive CD4+ T cells were resuspended in X-VIVO 15 and the cell density was adjusted to 400,000 cells / ml.

[0260] Add 50 μl diluted mDC, 100 μl naive CD4+ T cells, and 50 μl antibody to a 96-well U-plate with ultra-low adsorption. Incubate the 96-well U-plate at 37°C in a 5% CO2 cell culture incubator for 4 days. After 4 days of co-incubation, remove the 96-well U-plate, centrifuge at 500×g for 10 min, and discard 140 μl of supernatant. Dilute the 4× antibody as above, and add 120 μl of fresh X-VIVO 15 medium and 40 μl of 4× antibody to the 96-well U-plate with ultra-low adsorption. Mix the cells in the 96-well U-plate with a retort. Incubate the 96-well U-plate at 37°C in a 5% CO2 cell culture incubator for another 3 days. Remove the ultra-low adsorption 96-well U-plate, centrifuge at 500×g for 10 min, and discard 150 μl of supernatant. Dilute Anti-Human CD28 to 3.33 μg / ml (1.67×) with X-VIVO 15, and add 120 μl to each well of the U-plate coated with OKT3 the previous day. Dilute the 4× antibody as above, and add 40 μl of antibody to each well of the U-plate coated the previous day. Transfer all cells from the centrifuged 96-well U-plate to the previously coated 96-well U-plate. Incubate at 37°C for 1 day. Remove the 96-well U-plate from the incubator, centrifuge at 500×g for 10 min, and transfer 140 μl of supernatant to a new V-plate. Place the plate at -80℃ for ELISA experiments. Use the Human IL-5 DuoSet ELISA and Human IL-13 ELISA BASIC kit (HRP) to detect cytokines.

[0261] Figure 3 shows the experimental results. As can be seen from Figure 3, the blocking antibody against OX40L can significantly inhibit the Th2 inflammatory response.

[0262] 1.5 Role of anti-OX40L antibody in graft-versus-host disease (GvHD) mouse model

[0263] Part 1: Animal Experimentation and Disease Scoring for GvHD

[0264] NOG mice were intravenously injected with PBMCs (1*10^7 / mouse) to establish a GVHD model. Immediately, the mice were randomly divided into four groups of nine mice each. The drugs were administered weekly for four weeks, on days 0, 7, 14, and 21. Mouse weight was recorded twice weekly to observe survival, and weight change graphs and survival curves were generated (Figure 4). Blood samples were collected on day 14 for flow cytometry analysis. In this experiment, HZ22 effectively reduced weight loss caused by GVHD in mice and significantly reduced mortality; HZ18 significantly reduced mortality.

[0265] PBMC Information

[0266] Manufacturer: AllCellS Part Number: FPB005F-C Lot Number: 3024702

[0267] Mouse information

[0268] Strain: NOG Sex: Female Source: Vital River Laboratory Animal Technology Co., Ltd. Certificate No.: 110011211111216336

[0269] Antibody information

[0270] Part Two: Markers Related to Streaming Detection

[0271] In this experiment, blood was collected on day 14 of modeling, and the number of CD4 T cells in mouse blood was analyzed by flow cytometry. The proportion of Granzyme B-positive cells in CD4 T cells was also detected. Simultaneously, the levels of IFN-γ and IL-6 in serum were detected by ELISA. As shown in Figure 5, the blocking antibody OX40L can inhibit the secretion of IFN-γ and IL-6 in the peripheral blood of GvHD mice, and also inhibit T cell activation.

[0272] Example 2. Development and preparation of HZ22 protein solution formulation

[0273] To obtain a solution formulation that allows for long-term stable storage of HZ22 protein and ensures product quality within its shelf life, pre-formulation studies, formulation screening studies, and formulation determination studies were designed to finalize the HZ22 formulation. The HZ22 solution formulation is an injectable drug, administered via subcutaneous injection or intravenous infusion. To meet clinical subcutaneous administration requirements, the HZ22 concentration was increased, resulting in a clinical batch specification of 300 mg (2 ml) / vial.

[0274] 2.1. Pre-prescription research

[0275] 2.1.1 Research Plan

[0276] This study primarily investigated the effect of different pH values ​​on protein stability. Buffer solutions containing 1.55 mg / ml histidine and 50.00 mg / ml sorbitol were prepared, and the pH was adjusted to 5.0, 5.5, 6.0, 6.5, and 7.0 with hydrochloric acid. HZ22 protein was ultrafiltered and transferred to these buffer solutions at different pH values ​​to adjust the protein concentration to approximately 150 mg / ml. Polysorbate 80 was added to achieve a final concentration of 0.5 mg / ml. The samples were filtered, dispensed into vials, and capped. The stability of these samples was investigated at 40℃ ± 2℃, as detailed in Table 2.

[0277] Table 2. Research Plan Note: (1) √ indicates that the sample was taken at that point. (2) After the samples were taken at the above time points, they were first frozen at -70℃ and then thawed and sent for testing as needed.

[0278] The detection methods used in this experiment are as follows:

[0279] Visible foreign body observation method: Performed according to General Chapter 0904, Part IV, Chinese Pharmacopoeia 2020 Edition.

[0280] UV method: After the sample is thoroughly mixed, 2.5 μL is added to the chip, and the protein content is determined by a micro-ultraviolet spectrophotometer.

[0281] SEC-HPLC method: Prepare the mobile phase (20 mmol / L phosphate buffer + 400 mmol / L NaClO4, pH 6.8). Dilute the sample to 2.0 mg / ml with the mobile phase. The blank sample is ultrapure water. Connect the column and set the chromatographic parameters: flow rate: 0.5 ml / min; acquisition time: 30 min; injection volume: 50 μl; column temperature: 25℃; detection wavelength: 280 nm; sample pan temperature: 10℃. Integrate the chromatographic peak from 9.0 to 22.5 min, and calculate the protein purity using the peak area normalization method.

[0282] Non-reducing CE-SDS method: Prepare sample dilution buffer (pipette 200 μl of pH 6.5 citrate-phosphate buffer and add 80 μl of 10% SDS, then add water to bring the volume to 1 ml). Dilute the sample to 2 mg / ml. The blank sample is ultrapure water. Take 50 μl of sample and add 45 μl of pH 6.5 sample buffer and 5 μl of 250 mmol / L... After thorough mixing, NEM was heated at 70°C for 10 minutes. After cooling to room temperature, the sample was transferred to a sample tube for analysis. High-resolution separation mode was used, with injection from the left end of the capillary; the effective separation length of the capillary was 20.2 cm. Sample injection was performed using voltage: -5 kV for 40 seconds; the separation voltage was -15 kV for 35 minutes, and the Autozero time was 5 minutes. Integration parameters were used: peak width 0–200, threshold 0–100. Protein purity was calculated using peak area normalization. The percentage of the corrected peak area of ​​the main peak for non-reduced sample protein purity was reported, along with fragment content.

[0283] Charge Variant Assay (iCIEF method): Dilute the sample to 1.0 mg / ml with ultrapure water. Take 20 μl of the diluted sample and mix it thoroughly with 70 μl of 3 mol / L urea-0.5% MC solution, 4 μl of Pharmalyte 3-10, 2 μl of 500 mmol / L arginine, 0.3 μl of pI 5.41 marker and 0.3 μl of pI 9.91 marker. Centrifuge at 13000 rpm for 3 minutes, transfer to a 96-well plate, centrifuge at 3000 rpm for 5 minutes, and then inject for detection. Set the integration parameters, calculate the content of the principal component, acidic component and basic component according to the area normalization method, and report the pI of the main peak.

[0284] 2.1.2 Research Results

[0285] The results of the pre-prescription study are shown in Table 3 and Figures 6 (A to D).

[0286] Table 3. Results of Pre-Prescription Studies

[0287] The results showed that after 4 weeks of storage at 40℃±2℃, all samples at various pH levels were colorless and clear liquids without visible foreign matter, and the protein content and pH value did not change significantly (protein content changes were ≤10%, and pH value changes were ≤0.3). For purity determined by SEC-HPLC, the purity of samples at pH 5.5 and pH 6.0 decreased more slowly than that of samples at other pH conditions (Figure 1, A), decreasing by 1.7% and 1.8% respectively over four weeks; the pH 5.0 sample decreased the second most. For purity determined by non-reducing CE-SDS, the purity of the pH 5.5 sample decreased the slowest, decreasing by only 2.9% over four weeks (Figure 1, B). For charge variants—acidic components and principal components—the changes in pH 5.0 and pH 5.5 samples were more gradual, followed by the pH 6.0 sample (Figure 1, C and D).

[0288] The results of purity and charge variants indicate that the HZ22 protein has good stability at pH 5.0 to 6.0, and pH 5.5 was ultimately determined as the optimal pH for formulation determination studies.

[0289] 2.2. Prescription Screening Study

[0290] 2.2.1 Research Plan

[0291] This study primarily investigated the effects of excipients (sorbitol, arginine hydrochloride, and disodium edetate) on the stability of HZ22 protein. Detailed formulation information is shown in Table 4. Buffer solutions containing all components except HZ22 protein and polysorbate 80 were prepared according to Table 4. HZ22 protein was then ultrafiltered and displaced into the respective formulation solutions. After displacement, the protein concentration of each formulation was adjusted to 100 mg / ml. Polysorbate 80 was added to a final concentration of 0.5 mg / ml. The solutions were filtered, dispensed into vials, and capped. The stability of the samples was investigated at 40℃±2℃, as detailed in Table 5.

[0292] Table 4. Prescription Information Note: Adjust the pH with hydrochloric acid.

[0293] Table 5. Stability Testing Scheme Note: (1) √ indicates that the sample was taken at that point. (2) After the samples were taken at the above time points, they were first frozen at -70℃ and stored for testing. They were then thawed and sent for testing as needed.

[0294] The detection method used in this experiment is as follows:

[0295] Binding activity (cell ELISA method): OX40L cell suspension was seeded at a density of 0.4*10E6 cells / ml, 100 μL / well, and incubated for 2-20 h. After incubation, cells were fixed with 4% paraformaldehyde and blocked with 2% BSA-PBST. After blocking for 2 h, the plates were washed with 0.05% PBST, and serially diluted samples and reference standards were added. The plates were incubated at 37°C for 1 h. After sample incubation, the plates were washed, and detection antibodies were added and incubated for 30 min. After incubation, the plates were washed again, and TMB was added for color development for 15 min. The color development was terminated with ELISA stop solution. Signal values ​​at 450 nm and 620 nm were read using a microplate reader. A four-parameter curve was fitted with sample concentration on the x-axis and OD value on the y-axis to obtain the EC50 value and calculate the relative binding activity.

[0296] Polysorbate 80 content (UHPLC-CAD): Prepare mobile phase A: 2% formic acid aqueous solution (measure 20 ml of formic acid with a graduated cylinder, add 980 ml of ultrapure water, and mix well), and prepare mobile phase B: isopropanol. Prepare a 2.5% PS80 stock solution (accurately weigh 1.25 g of PS80, add 48.75 g of ultrapure water, and mix for 30 minutes until fully dissolved), prepare a 2.0 mg / ml PS solution (take 120 μl of 2.5% PS stock solution, add 1380 μl of ultrapure water, and mix well), prepare a standard solution STD (take 2.0 mg / ml PS, and dilute with ultrapure water to 0.05 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.50 mg / ml, 0.75 mg / ml, and 1.00 mg / ml, respectively), and a quality control solution STD-0.40 (take 40 μl of 2.5% PS stock solution, add 960 μl of ultrapure water to dilute to 1.00 mg / ml, mix well, and then take 400 μl of this solution and add 600 μl of ultrapure water to further dilute to 0.40 mg / ml). Prepare the test solution (ultrapure water and sample are thoroughly mixed 1:1 (v / v) before analysis). Power on the CAD and purge with nitrogen. Allow the detector to equilibrate for 30 minutes before the mobile phase passes through it to ensure pressure stability. Flush the system with 100% mobile phase B at a rate of 1.0 ml / min for approximately 15 minutes. Then flush the system with 90% mobile phase A at a rate of 1.0 ml / min for approximately 15 minutes. Connect the column and set the method parameters: evaporator temperature: 50℃; flow rate: 1.0 ml / min; acquisition time: 10 min; injection volume: 15 μl; column temperature: 35℃. After equilibrating the column to a stable baseline with the mobile phase, equilibrate with sample water first. Once the observed chromatograms are consistent and free of interference, begin the analysis. If interfering peaks are present, replace the mobile phase or column. In the sequence parameter table, the 1:1 (v / v) dilution ratio must be considered for the test sample injection; when writing the sequence, the dilution factor of 2 must be clearly stated. Based on the results of STD-1 at concentrations of 0.05 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.50 mg / ml, 0.75 mg / ml, and 1.00 mg / ml, a standard curve was fitted using a quadratic function with the peak area and corresponding PS concentration for each concentration. The standard curve does not need to cross zero. The reference formula for the quadratic function fitted standard curve between the PS peak area and the corresponding PS concentration (mg / ml) is: Y = Ax² + Bx + C; where: Y is the peak area of ​​the main PS peak in the sample; x is the PS concentration in the sample; A is the binomial coefficient, determining the curve type; B is the slope of the standard curve; and C is the Y-intercept. The preferred polysorbate 80 content is in the range of 0.2-0.8 mg / ml.

[0297] Other detection methods are as described above.

[0298] 2.2.2 Research Results

[0299] The results of the prescription screening study are shown in Table 6 and Figure 7 (A to D).

[0300] Table 6. Results of Prescription Screening Study Note: "N / A" indicates that the sample was not submitted for testing.

[0301] The results showed that after 4 weeks of storage at 40℃±2℃, all formulation samples met the appearance requirements (appearing as colorless, clear liquids or slightly opalescent), with no visible foreign matter, and no significant changes in protein content or pH value (protein content changes ≤10%, pH value changes ≤0.3). The stability trends (purity, charge variants) of formulations 1, 2, and 3 showed no significant differences, while the stability trend of formulation 4 was more moderate. The binding activity of all formulations was within acceptable limits. Formulation 4 showed the least reduction in polysorbate 80 content.

[0302] Since the stability trends of formulations 1, 2, and 3 showed no significant difference, formulation 1 was selected as the preferred 100 mg / ml formulation for HZ22 for the purpose of simplicity. Formulation 4, compared to formulation 3, added 0.01 mg / ml disodium edetate, resulting in a more moderate decrease in polysorbate 80 content and purity; therefore, 0.02 mg / ml disodium edetate was added. The sorbitol content was adjusted to 50.00 mg / ml to regulate osmotic pressure. To avoid using hydrochloric acid to adjust pH during production, the buffer system was adjusted to histidine and histidine hydrochloride. Based on the above results, the preferred 100 mg / ml formulation for HZ22 is: 100.0 mg / ml anti-OX40L antibody, 0.77 mg / ml histidine, 3.40 mg / ml histidine hydrochloride, 50.00 mg / ml sorbitol, 0.02 mg / ml disodium edetate, and 0.5 mg / ml polysorbate 80.

[0303] 2.3. Prescription Determination Research

[0304] 2.3.1 Research Plan

[0305] This study primarily investigated the effect of excipients (sorbitol, arginine hydrochloride, sodium chloride, and disodium edetate) on the stability of HZ22 protein after increasing the protein concentration to 150 mg / ml. Detailed formulation information is shown in Table 7. Buffer solutions containing all components except HZ22 protein and polysorbate 80 were prepared according to Table 7. HZ22 protein was then ultrafiltered and displaced into the respective formulation solutions. After displacement, the protein concentration of each formulation was adjusted to 150 mg / ml. Polysorbate 80 was added to a final concentration of 0.5 mg / ml. The solutions were filtered, dispensed into vials, and capped. The stability of the samples was investigated at 40℃±2℃, as detailed in Table 8. The detection methods are as described above.

[0306] Table 7. Prescription Information Note: Adjust the pH with hydrochloric acid.

[0307] Table 8. Stability Testing Scheme Note: (1) √ indicates that the sample was taken at that point. (2) After the samples were taken at the above time points, they were first frozen at -70℃ and stored for testing. They were then thawed and sent for testing as needed.

[0308] 2.3.2 Research Results

[0309] The results of the prescription determination study are shown in Table 9 and Figure 8 (A to D).

[0310] Table 9. Results of the Prescription Determination Study Note: N / A indicates that it was not submitted for testing.

[0311] The results showed that after 4 weeks of storage at 40℃±2℃, the samples of all formulations were aesthetically pleasing (appearing as colorless and clear liquids), free of visible foreign matter, and exhibited no significant changes in protein content or pH (protein content changes were ≤10%, and pH changes were ≤0.3). The purity and charge variant changes of all formulations were within acceptable ranges, with formulation 5 showing a more moderate decrease in purity (SEC-HPLC method and non-reducing CE-SDS method). The polysorbate 80 content of all formulation samples remained unchanged. The binding activity of formulation 5 was within acceptable limits.

[0312] Based on the results of pre-formulation studies, formulation screening, and formulation determination studies, formulation 5 was selected as the optimal formulation for HZ22. To avoid using hydrochloric acid to adjust the pH during production, the buffer system was adjusted to histidine and histidine hydrochloride. Therefore, the final formulation for the HZ22 clinical batch is: 150.0 mg / ml anti-OX40L antibody, 0.77 mg / ml histidine, 3.40 mg / ml histidine hydrochloride or 3.44 mg / ml histidine hydrochloride, 40.00 mg / ml sorbitol, 0.02 mg / ml disodium edetate, and 0.5 mg / ml polysorbate 80, pH 5.7. It is stable for at least 12 months, e.g., 24 months, under storage conditions (e.g., 2–8°C or ambient temperature).

[0313] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0314] Sequence information

Claims

1. A solution formulation comprising an anti-OX40L antibody or an antigen-binding fragment thereof, the solution formulation having a pH of about 5.0 to about 8.0; wherein the OX40L antibody or the antigen-binding fragment thereof comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), 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 or are composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7; Optionally, the anti-OX40L antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein (i) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence; (ii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8; or (iii) The heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:

8.

2. The solution formulation according to claim 1, wherein the anti-OX40L antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4, preferably from human IgG1, IgG2, IgG3, or IgG4. Optionally, the heavy chain constant region contains mutations that improve antibody stability, such as mutations that eliminate the heterogeneity of IgG. For example, the S228P (EU number) mutation; Optionally, the heavy chain constant region (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:13; (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 shown in SEQ ID NO:11, and having an S228P mutation; or It contains an amino acid sequence selected from or composed of the amino acid sequence shown in SEQ ID NO: 11 or 13; Optionally, the antibody light chain constant region is a light chain constant region derived from the human lambda or Kappa light chain constant region, preferably the human lambda light chain constant region or the human Kappa light chain constant region; Optionally, the light chain constant region (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.

3. The solution formulation according to any one of the preceding claims, wherein the anti-OX40L antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein (i) The heavy chain comprises at least 85%, 90%, 91%, and 92% of the amino acid sequence shown in SEQ ID NO:

9. The light chain comprises or consists 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:10; and the light chain comprises or consists 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:

10. (ii) The heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:9, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:10; or (iii) The heavy chain consists of the amino acid sequence shown in SEQ ID NO:9, and the light chain consists of the amino acid sequence shown in SEQ ID NO:

10.

4. The solution formulation according to any one of the preceding claims, wherein the anti-OX40L antibody or its antigen-binding fragment may be present at a concentration of about 1-500 mg / mL, for example about 10-400 mg / mL, about 20-300 mg / mL, about 40-200 mg / mL, about 50-150 mg / mL, or about 100-150 mg / mL, preferably about 100 mg / mL or about 150 mg / mL.

5. The solution formulation according to any one of the preceding claims, wherein the solution formulation has a pH of about 5.0-7.4, such as about 5.0-7.0, about 5.0-6.0, about 5.5-6.0, about 5.5-5.7, such as about 5.5, about 5.6, or about 5.

7.

6. The solution formulation according to any one of the preceding claims, comprising a buffer, optionally the buffer comprising one or more selected from: acetate, such as sodium acetate, potassium acetate; tartaric acid and / or its salts, such as sodium tartrate, potassium tartrate; carbonate, bicarbonate, such as sodium carbonate, sodium bicarbonate; citric acid and / or its salts, such as sodium citrate, potassium citrate; amino acids and / or their salts, such as amino acids and / or their hydrochloride or sodium salts; phosphates, such as sodium phosphate; hydrogen phosphates, such as sodium dihydrogen phosphate, disodium hydrogen phosphate; Tris (tris(hydroxymethyl)aminomethane); HEPES (4-hydroxyethylpiperazine ethanesulfonic acid); MES buffer; and MOPS buffer; Optionally, the buffer comprises an amino acid and / or its salt, such as an amino acid and / or its hydrochloride or sodium salt, preferably an amino acid and / or its hydrochloride; preferably, the buffer comprises one or more selected from histidine, histidine hydrochloride, arginine, arginine hydrochloride, glycine, lysine, and monosodium glutamate; more preferably, the buffer comprises one or more selected from histidine, histidine hydrochloride, arginine, and arginine hydrochloride. Optionally, the buffer comprises histidine and / or histidine hydrochloride, for example, the buffer is histidine, or the buffer is histidine hydrochloride, or the buffer is a combination of histidine and histidine hydrochloride, wherein the weight ratio of histidine to histidine hydrochloride is optionally about 1:2 to 1:10, preferably about 1:3 to 1:6, more preferably about 1:4 to 1:5, for example about 1:4.42 or about 1:4.

46.

7. The solution formulation according to any one of the preceding claims, wherein... The solution formulation contains about 0.1-10 mg / mL of histidine, for example about 0.5-5 mg / mL, for example about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 mg / mL or any combination thereof. or The solution formulation comprises about 0.1-5 mg / mL histidine and about 0.4-25 mg / mL histidine hydrochloride. Preferably, the concentration of histidine is about 0.2-4 mg / mL, for example about 0.4-3 mg / mL, about 0.6-2 mg / mL, or about 0.7-0.8 mg / mL, and the concentration of histidine hydrochloride is about 0.5-20 mg / mL, for example about 1-15 mg / mL, about 2-10 mg / mL, or about 3-4 mg / mL. or The solution formulation comprises a combination of histidine and histidine hydrochloride at molar concentrations of about 2-200 mM, about 3-100 mM, about 4-50 mM, about 5-30 mM, and about 10-25 mM, based on histidine; preferably, the weight ratio of histidine to histidine hydrochloride is about 1:2 to 1:10, more preferably about 1:3 to 1:6, more preferably about 1:4 to 1:5, for example about 1:4.42 or about 1:4.

46.

8. The solution formulation according to any one of the preceding claims, comprising a surfactant, preferably a nonionic surfactant, such as sorbitan fatty acid esters, including polysorbate and Span; polypropylene glycol ethylene oxide adducts, including poloxamer; and polyoxyethylene alkyl ethers, including benzyl ether. Preferably, the surfactant is a polysorbate, such as polysorbate 20 or polysorbate 80, optionally at a concentration of about 0.01-10 mg / ml, such as about 0.05-6 mg / ml, about 0.1-3 mg / ml, about 0.2-2 mg / ml, about 0.3-1 mg / ml, about 0.4-0.6 mg / ml, such as about 0.5 mg / ml.

9. The solution formulation according to any one of the preceding claims, comprising an osmotic pressure regulator; Optionally, the osmotic pressure regulator is selected from one or more of sorbitol, xylitol, mannitol, fructose, glucose, sucrose, mannose, trehalose, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, ammonium sulfate, sodium nitrate, potassium nitrate, phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and calcium phosphate. Optionally, the osmotic pressure regulator comprises one or more of sorbitol, xylitol or mannitol, preferably sorbitol, optionally at a concentration of about 1-200 mg / ml, such as about 5-150 mg / ml, about 10-100 mg / ml, or about 20-60 mg / ml, such as about 40 mg / mL, such as about 50 mg / mL; Alternatively, the osmotic pressure regulator may comprise sodium chloride, optionally at a concentration of about 1-100 mg / ml, such as about 2-50 mg / ml, 2-20 mg / ml, 2-10 mg / ml, or 3-5 mg / ml.

10. The solution formulation according to any one of the preceding claims, comprising a protein stabilizer, said protein stabilizer optionally selected from one or more of the following: (1) Sugars and polyols, such as sucrose, lactose, trehalose, maltose, fructose, inulin oligosaccharides, dextran, glucose, maltodextrin, cyclodextrin, dextran, glycerol, mannitol, sorbitol, xylitol, ethylene glycol, etc. (2) Amino acids or their salts, such as arginine or its salts, glycine, sodium aspartate, glutamic acid, lysine, etc. (3) Complexing agents, such as edetic acid or its salts; (4) Surfactants, including ionic or nonionic surfactants, preferably nonionic surfactants; (5) Salts, such as sodium chloride, potassium chloride, etc.; (6) Polyethylene glycols, such as PEG200, PEG300, PEG400, PEG600, PEG1000, etc.; (7) Proteins, such as albumin, casein, mucopolysaccharide proteins, etc.; (8) Cyclodextrins, such as hydroxypropyl β-cyclodextrin, etc. (9) Other macromolecular compounds, such as heparin, hydroxyethyl starch, polyvinylpyrrolidone, etc. Preferably, the protein stabilizer is selected from one or more of the following: sorbitol, arginine or its hydrochloride, edetate or its salt, polysorbate and sodium chloride.

11. The solution formulation according to any one of the preceding claims, comprising a complexing agent, such as edemaic acid (EDTA) or a salt thereof, such as a sodium salt, for example, disodium edema or sodium calcium edema; optionally, the concentration of edemaic acid or a salt thereof in the solution formulation is about 0.005-0.05 mg / ml, for example 0.005-0.03 mg / ml, for example about 0.01-0.02 mg / ml, for example about 0.01 mg / ml or about 0.02 mg / ml.

12. The solution formulation according to any one of the preceding claims, comprising arginine and / or its salts, such as arginine and / or its hydrochloride; optionally, the concentration of arginine and / or its salts, such as hydrochloride, in the solution formulation is about 0.1-100 mg / mL, for example about 1-80 mg / mL, about 5-50 mg / mL, or about 15-35 mg / mL.

13. The solution formulation according to any one of the preceding claims, comprising, or composed of, the following components: (a) The anti-OX40L antibody or its antigen-binding fragment; (b) A buffer, preferably comprising an amino acid and / or its hydrochloride salt, more preferably comprising histidine and / or histidine hydrochloride; (c) Surfactant, preferably nonionic surfactant, more preferably polysorbate; (d) Optionally, the osmotic pressure regulator is preferably a polyol (e.g., sorbitol, xylitol or mannitol) or sodium chloride; (e) Optionally, a complexing agent, such as edetate and / or its salts (e.g., sodium salt); (f) Optionally, arginine and / or its hydrochloride; (g) Solvent, preferably water; The solution formulation has a pH of about 5.0-8.0, preferably about 5.0-7.4, such as about 5.0-7.0, about 5.0-6.0, about 5.5-6.0, about 5.5-5.7, such as about 5.5, about 5.6 or about 5.

7.

14. The solution formulation according to any one of the preceding claims, comprising, or composed of, the following components: (a) The anti-OX40L antibody or its antigen-binding fragment, optionally at a concentration of about 1-500 mg / mL; (b) Histidine and / or histidine hydrochloride, optionally at a concentration of about 0.1-100 mg / mL, for example about 0.5-60 mg / mL, about 1-30 mg / mL, or about 1-10 mg / mL; or at a concentration of about 1-500 mM, for example about 2-200 mM, about 3-100 mM, about 4-50 mM, about 5-30 mM, or about 10-25 mM, wherein the weight ratio of said histidine to histidine hydrochloride is optionally about 1:2 to 1:10, preferably about 1:3 to 1:6, more preferably about 1:4 to 1:5, for example about 1:4.42 or about 1:4.46; (c) Polysorbate, optionally at a concentration of about 0.01-10 mg / ml, for example about 0.05-6 mg / ml, about 0.1-3 mg / ml, about 0.2-2 mg / ml, about 0.3-1 mg / ml, about 0.4-0.6 mg / ml; (d) Optionally, a polyol (e.g., sorbitol, xylitol or mannitol), preferably sorbitol, is used at a concentration of about 1-200 mg / ml, for example, about 5-150 mg / ml, about 10-100 mg / ml or about 20-60 mg / ml; (e) Optionally, edetate and / or its sodium salt, such as disodium edetate or calcium sodium edetate, at a concentration of about 0.005-0.05 mg / ml, such as about 0.005-0.03 mg / ml, such as about 0.01-0.02 mg / ml; (f) Optionally, sodium chloride, optionally at a concentration of about 1-100 mg / ml, for example about 2-50 mg / ml, 2-20 mg / ml, 2-10 mg / ml, or 3-5 mg / ml; (g) Optionally, arginine and / or its hydrochloride, optionally at a concentration of about 0.1-100 mg / mL, for example about 1-80 mg / mL, about 5-50 mg / mL, about 15-35 mg / mL; (h) Solvent, preferably water; The solution formulation has a pH of about 5.0-6.0, for example, about 5.5, about 5.6 or about 5.

7.

15. The solution formulation according to any one of the preceding claims, comprising, or consisting of, the following components. composition: (a) Approximately 100-150 mg / mL of anti-OX40L antibody or its antigen-binding fragment; (b) Approximately 0.5-5 mg / mL of histidine, or a combination of approximately 0.7-0.8 mg / mL of histidine and approximately 3-4 mg / mL of histidine hydrochloride; Alternatively, based on the concentration of histidine, approximately 5-30 mM or approximately 10-25 mM of histidine and / or histidine hydrochloride, optionally with a weight ratio of histidine to histidine hydrochloride of approximately 1:2 to 1:10, preferably approximately 1:3 to 1:6, more preferably approximately 1:4 to 1:5, for example approximately 1:4.42 or approximately 1:4.46; (c) Polysorbate 80, optionally at a concentration of about 0.4-0.6 mg / ml; (d) Polyols (e.g., sorbitol, xylitol or mannitol), preferably sorbitol, optionally at a concentration of about 20-60 mg / ml; (e) Edesmonic acid and / or its sodium salt, such as disodium edetate or calcium sodium edetate, optionally at a concentration of about 0.01-0.02 mg / ml; (f) Optionally, sodium chloride, optionally at a concentration of about 3-5 mg / ml; (g) Optionally, arginine and / or its hydrochloride, optionally at a concentration of about 15-35 mg / ml; (h) Solvent, preferably water; The solution formulation has a pH of about 5.0-6.0, for example, about 5.5, about 5.6 or about 5.

7. Alternatively, the solution formulation comprises, or is composed of, the following components. composition: (a) Approximately 100 mg / mL of anti-OX40L antibody or its antigen-binding fragment; (b) about 0.77 mg / ml of histidine and about 3.40 mg / ml of histidine hydrochloride, or about 0.77 mg / ml of histidine and about 3.44 mg / ml of histidine hydrochloride; or a combination of histidine and histidine hydrochloride, wherein the concentration of histidine in the solution is about 20 mM, 21 mM or 22 mM, optionally the weight ratio of said histidine to histidine hydrochloride is about 1: 4.42 or approximately 1:4.46; (c) Approximately 0.5 mg / mL of polysorbate 80; (d) Approximately 50 mg / mL of sorbitol; (e) Sodium edetate at approximately 0.02 mg / ml; (f) Solvent, preferably water; The solution formulation has a pH of about 5.0-8.0, preferably about 5.0-6.0, for example about 5.5, 5.6 or 5.7; Alternatively, the solution formulation may comprise, or be composed of, the following components: (a) Approximately 150 mg / mL of anti-OX40L antibody or its antigen-binding fragment; (b) about 0.77 mg / ml of histidine and about 3.40 mg / ml of histidine hydrochloride; or about 0.77 mg / ml of histidine and about 3.44 mg / ml of histidine hydrochloride; or a combination of histidine and histidine hydrochloride, wherein the concentration of histidine in the solution is about 20 mM, 21 mM or 22 mM, optionally the weight ratio of said histidine to histidine hydrochloride is about 1: 4.42 or approximately 1:4.46; (c) Approximately 40 mg / mL of sorbitol; (d) Sodium edetate at approximately 0.02 mg / ml; (e) Approximately 0.5 mg / mL of polysorbate 80; (f) Solvent, preferably water; The solution preparation has a pH of about 5.0-8.0, preferably about 5.0-6.0, for example about 5.5, 5.6 or 5.

7.

16. The solution preparation according to any one of claims 1-15, wherein it is an injectable preparation.

17. A pre-filled syringe containing a solution formulation according to any one of claims 1-16, for example for intravenous or subcutaneous injection, such as subcutaneous bolus injection.

18. A solid formulation obtained by solidifying the solution formulation according to any one of claims 1-16, for example by freeze-drying or spray drying.

19. A solution formulation according to any one of claims 1-16, a pre-filled syringe according to claim 17, or a solid formulation according to claim 18 for the prevention or treatment of diseases or conditions related to abnormal activation of the OX40L / OX40-mediated pathway; optionally, the subject suffering from the disease has abnormal activation of the OX40L / OX40-mediated signaling pathway compared to a healthy individual; optionally, the disease is selected from autoimmune diseases or allogeneic transplantation, for example, the autoimmune diseases include, but are not limited to, allergic dermatitis, asthma, systemic lupus erythematosus, Schogren's syndrome, immune thrombocytopenic purpura, multiple sclerosis, lupus nephritis, amyotrophic lateral sclerosis, rheumatoid arthritis, non-rheumatoid arthritis, contact dermatitis, hyperIgE syndrome, inflammatory bowel disease, myasthenia gravis, Greaves' disease, hemolytic anemia, psoriasis, atopic dermatitis, allergic asthma, or idiopathic inflammatory disease.

20. Use of the solution formulation of any one of claims 1-16, the pre-filled syringe of claim 17, or the solid formulation of claim 18 for the prevention or treatment of diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway, or for the preparation of a medicament for the prevention or treatment of diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway; optionally, the subject suffering from said disease has abnormal activation of the OX40L / OX40-mediated signaling pathway compared to a healthy individual; Optionally, the disease is selected from autoimmune diseases or allogeneic transplantation. For example, the autoimmune diseases include, but are not limited to, allergic dermatitis, asthma, systemic lupus erythematosus, Schogren's syndrome, immune thrombocytopenic purpura, multiple sclerosis, lupus nephritis, amyotrophic lateral sclerosis, rheumatoid arthritis, non-rheumatoid arthritis, contact dermatitis, hyperIgE syndrome, inflammatory bowel disease, myasthenia gravis, Greaves' disease, hemolytic anemia, psoriasis, atopic dermatitis, allergic asthma, or idiopathic inflammatory diseases.

21. A method for preventing or treating diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway in an individual, the method comprising administering, for example by injection, an effective amount of the solution formulation of any one of claims 1-16, the pre-filled syringe of claim 17, or the solid formulation of claim 18 to the individual; optionally, the subject suffering from the disease has abnormal activation of the OX40L / OX40-mediated signaling pathway compared to a healthy individual; optionally, the disease is selected from autoimmune diseases or allogeneic transplantation, for example, the autoimmune diseases include, but are not limited to, allergic dermatitis, asthma, systemic lupus erythematosus, Schogren's syndrome, immune thrombocytopenic purpura, multiple sclerosis, lupus nephritis, amyotrophic lateral sclerosis, rheumatoid arthritis, non-rheumatoid arthritis, contact dermatitis, hyperIgE syndrome, inflammatory bowel disease, myasthenia gravis, Greaves' disease, hemolytic anemia, psoriasis, atopic dermatitis, allergic asthma, or idiopathic inflammatory disease.

22. A kit comprising a solution formulation according to any one of claims 1-16, a pre-filled syringe according to claim 17, or a solid formulation according to claim 18, and instructions for use of said formulation for the prevention or treatment of diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway; optionally, when said formulation is a solid formulation, the kit further comprises a reconstituted solvent such as water in a separate container; and / or optionally, a subject suffering from said disease, compared to a healthy individual, has an OX40L / OX40-mediated signaling pathway. Abnormal activation; optionally, the disease is selected from autoimmune diseases or allogeneic transplantation, for example, the autoimmune diseases include but are not limited to allergic dermatitis, asthma, systemic lupus erythematosus, Schoenberg syndrome, immune thrombocytopenic purpura, multiple sclerosis, lupus nephritis, amyotrophic lateral sclerosis, rheumatoid arthritis, non-rheumatoid arthritis, contact dermatitis, hyperIgE syndrome, inflammatory bowel disease, myasthenia gravis, Greaves' disease, hemolytic anemia, psoriasis, atopic dermatitis, allergic asthma or idiopathic inflammatory disease.

23. A combination of the solution formulation of any one of claims 1-16, the pre-filled syringe of claim 17, or the solid formulation of claim 18 with one or more other therapies, such as pharmacological treatment and / or surgical treatment, for example, for the prevention or treatment of diseases or conditions associated with abnormal activation of the OX40L / OX40-mediated pathway; optionally, a subject suffering from said disease has abnormal activation of the OX40L / OX40-mediated signaling pathway compared to a healthy individual; optionally, said disease is selected from autoimmune diseases or allogeneic transplantation, for example, said autoimmune diseases include, but are not limited to, allergic dermatitis, asthma, systemic lupus erythematosus, Schogren's syndrome, immune thrombocytopenic purpura, multiple sclerosis, lupus nephritis, amyotrophic lateral sclerosis, rheumatoid arthritis, non-rheumatoid arthritis, contact dermatitis, hyperIgE syndrome, inflammatory bowel disease, myasthenia gravis, Greaves' disease, hemolytic anemia, psoriasis, atopic dermatitis, allergic asthma, or idiopathic inflammatory disease.

24. A method for preparing a solution formulation according to any one of claims 1-16, the method comprising: (a) Dissolve the buffer in water to obtain a buffer solution, wherein the buffer solution preferably has the desired pH; (b) Add the OX40L antibody or its antigen-binding fragment to the buffer solution of step (a); (c) Add a surfactant to the solution from step (b) to obtain the solution formulation; (d) Optionally, the solution from step (c) is aseptically filtered; and (e) Optionally, the solution from step (d) is dispensed into vials and sealed to obtain the finished product; Optionally, when the solution formulation contains additional excipients, these excipients are added to the buffer solution in step (a).

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