Pharmaceutical composition containing Anti-GPC3 / CD3 bispecific antibody

Abstract

Provided is a pharmaceutical composition containing an anti-GPC3 and CD3 bispecific antigen binding molecule and an excipient. The pharmaceutical composition has an excellent antibody stability.

Description

Pharmaceutical composition containing anti-GPC3 / CD3 bispecific antibody

[0001] This application claims priority to the Chinese patent application No.CN2024104369894, filed on April 11, 2024, entitled "Pharmaceutical composition containing anti-GPC3 / CD3 bispecific antibody", the Chinese patent application No.CN2025104291767, filed on April 03, 2025, entitled "Pharmaceutical composition containing anti-GPC3 / CD3 bispecific antibody", the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0002] The present disclosure relates to the field of pharmaceutical formulations, in particular to a new pharmaceutical composition containing anti-GPC3 / CD3 bispecific antigen binding molecules. BACKGROUND

[0003] Glypican 3 (GPC3) is a kind of protein glycan attached to the surface of cells through glycerophosphatidylinositol (GPI) anchor, belonging to the heparan sulfate proteoglycan (HSPG) family, mainly involved in the regulation of cell proliferation, adhesion and migration and other processes. GPC3 is highly expressed in liver cancer tissue, present in 70%-80% of hepatocellular carcinoma (HCC) patients, while its expression in normal tissues of adults is very low or not expressed. Therefore, GPC3 has become an auxiliary diagnostic marker and therapeutic target for liver cancer.

[0004] Cluster of differentiation 3 (CD3) is a homodimer or heterodimer antigen expressed on T cells associated with T cell receptor complex (TCR), which is essential for T cell activation. It has been proved that the antibody against CD3 will aggregate CD3 on T cells, thus causing T cell activation in a similar way to the engagement of TCR by peptide-loaded MHC molecules, so the anti-CD3 antibody is involved in T cell activation therapy. Anti-GPC3 / CD3 antibody targets GPC3 antigen on the surface of human tumor cells through GPC3, and recruits T cells to the tumor site through CD3 target, so as to activate T cells to kill GPC3 positive tumor cells, and achieve the purpose of inhibiting the growth of tumor cells.

[0005] At the same time, therapeutic macromolecules (e.g., antibodies) should not only be able to make the molecule suitable for administration to a patient, but also should be able to maintain sufficient stability during storage and use. For example, a therapeutic antibody in solution is susceptible to degradation, aggregation, and / or undesirable chemical changes unless the solution is properly formulated. In addition, when preparing an antibody formulation, other factors need to be considered in addition to stability. Examples of such other considerations include the compatibility of the solution with commonly used clinical compounding solutions at the time of clinical administration, and the visual quality or appearance of the formulation. Thus, when formulating a therapeutic antibody, great care must be taken to obtain a formulation that remains stable, contains sufficient concentration of the antibody, and has other properties that are convenient for administration to a patient.

[0006] Therefore, there is still an urgent need in the art for a pharmaceutical composition of anti-GPC3 / CD3 bispecific antigen-binding molecule with higher activity, stable performance, and facilitating production and administration.

[0007] SUMMARY

[0008] The present disclosure provides a pharmaceutical composition containing an anti-GPC3 / CD3 bispecific antigen-binding molecule, which has good stability.

[0009] The present disclosure provides a pharmaceutical composition comprising an anti-GPC3 / CD3 bispecific antigen-binding molecule and an excipient, the excipient being optionally selected from one or more of a stabilizer and a solubilizer, wherein the anti-GPC3 / CD3 bispecific antigen-binding molecule comprises a first antigen-binding domain recognizing glypican 3 (GPC3), and a second antigen-binding domain recognizing a T cell receptor CD3 subunit; the first antigen-binding domain comprises a heavy chain variable region comprising HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, and HCDR3 as set forth in SEQ ID NO: 3; the second antigen-binding domain comprises a scFv comprising HCDR1 as set forth in SEQ ID NO: 4, HCDR2 as set forth in SEQ ID NO: 5, and HCDR3 as set forth in SEQ ID NO: 6, and LCDR1 as set forth in SEQ ID NO: 7, LCDR2 as set forth in SEQ ID NO: 8, and LCDR3 as set forth in SEQ ID NO: 9.

[0010] In some embodiments, the sequence of the heavy chain variable region of the first antigen-binding domain is set forth in SEQ ID NO: 10, and the sequence of the scFv of the second antigen-binding domain is set forth in SEQ ID NO: 11.

[0011] In some embodiments, the anti-GPC3 / CD3 bispecific antigen-binding molecules described in the present disclosure further have one or more of the following characteristics:

[0012] (1) the first antigen-binding domain is a nanobody; preferably, a humanized camelid nanobody;

[0013] (2) the bispecific antigen-binding molecule comprises a heavy chain constant region CH2 and CH3, and does not comprise a light chain constant region and / or a heavy chain constant region CH1; preferably, the heavy chain constant region comprises an Fc domain or a variant Fc; more preferably, the Fc is derived from murine or human;

[0014] (3) the bispecific antigen-binding molecule comprises an Fc domain, wherein the Fc domain is an IgG Fc domain; preferably, the Fc domain is an IgG1 domain or an IgG4 domain;

[0015] (4) the bispecific antigen-binding molecule comprises an Fc domain, wherein the two polypeptide chains constituting the Fc domain have different sequences from each other, and the amino acid residue at position 366 according to EU numbering in the Knob-Fc chain of the two polypeptides constituting the Fc domain is mutated to tryptophan, and the amino acid residue at position 366 according to EU numbering in the other Hole-Fc chain is mutated to serine, the amino acid residue at position 368 according to EU numbering is mutated to alanine, and the amino acid residue at position 407 according to EU numbering is mutated to valine; preferably, the Knob-Fc chain comprises S354C and T366W amino acid substitutions, and the Hole-Fc chain comprises Y349C, T366S, L368A and Y407V amino acid substitutions; more preferably, the Hole-Fc chain further comprises H435R substitution; further preferably, the Knob-Fc chain and / or the Hole-Fc chain further comprises L234A and L235A substitutions; optionally, the Knob-Fc chain and / or the Hole-Fc chain can further comprise K447A substitution at the C-terminal last position.

[0016] (5) the bispecific antigen-binding molecule comprises an Fc domain, wherein the Fc domain comprises a Knob-Fc chain and a Hole-Fc chain, the sequence of the Knob-Fc chain is shown in SEQ ID NO: 14, and the sequence of the Hole-Fc chain is shown in SEQ ID NO: 15.

[0017] In some embodiments, the anti-GPC3 / CD3 bispecific antigen-binding molecules described in the present disclosure comprise a first peptide chain and a second peptide chain, the sequence of the first peptide chain is shown in SEQ ID NO: 12, and / or the sequence of the second peptide chain is shown in SEQ ID NO: 13.

[0018] In some embodiments, the anti-GPC3 / CD3 bispecific antigen binding molecule is at a concentration of about 1 mg / mL to about 100 mg / mL; in some embodiments, the anti-GPC3 / CD3 bispecific antigen binding molecule is at a concentration of about 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 13 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, 23 mg / mL, 25 mg / mL, 28 mg / mL, 30 mg / mL, 33 mg / mL, 35 mg / mL, 38 mg / mL, 40 mg / mL, 43 mg / mL, 45 mg / mL, 48 mg / mL, 50 mg / mL, 53 mg / mL, 55 mg / mL, 58 mg / mL, 60 mg / mL, 63 mg / mL, 65 mg / mL, 68 mg / mL, 70 mg / mL, 73 mg / mL, 75 mg / mL, 78 mg / mL, 80 mg / mL, 83 mg / mL, 85 mg / mL, 88 mg / mL, 90 mg / mL, 93 mg / mL, 95 mg / mL, 98 mg / mL, 100 mg / mL, or any range between these point values.

[0019] In some embodiments, the stabilizer is selected from one or more of a sugar, an amino acid.

[0020] In some embodiments, the sugar is selected from the group consisting of conventional compositions (CH20)nand derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. The sugar can be selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, and the like. Preferably, the sugar is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol.

[0021] In some embodiments, the concentration of the sugar is about 20 mg / mL to about 100 mg / mL. In some embodiments, the concentration of the sugar is about 20 mg / mL, 23 mg / mL, 25 mg / mL, 28 mg / mL, 30 mg / mL, 33 mg / mL, 35 mg / mL, 38 mg / mL, 40 mg / mL, 43 mg / mL, 45 mg / mL, 48 mg / mL, 50 mg / mL, 53 mg / mL, 55 mg / mL, 58 mg / mL, 60 mg / mL, 63 mg / mL, 65 mg / mL, 68 mg / mL, 70 mg / mL, 73 mg / mL, 75 mg / mL, 78 mg / mL, 80 mg / mL, 83 mg / mL, 85 mg / mL, 88 mg / mL, 90 mg / mL, 93 mg / mL, 95 mg / mL, 98 mg / mL, 100 mg / mL, or any range between these point values.

[0022] In some embodiments, the amino acid is selected from proline or a pharmaceutically acceptable salt, methionine or a pharmaceutically acceptable salt, glycine or a pharmaceutically acceptable salt, phenylalanine or a pharmaceutically acceptable salt, valine or a pharmaceutically acceptable salt, leucine or a pharmaceutically acceptable salt.

[0023] In some embodiments, the concentration of the amino acid is about 0 to about 100 mM. In some embodiments, the concentration of the amino acid is about 0 mM, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 18 mM, 20 mM, 25 mM, 28 mM, 30 mM, 35 mM, 38 mM, 40 mM, 45 mM, 48 mM, 50 mM, 55 mM, 58 mM, 60 mM, 65 mM, 68 mM, 70 mM, 75 mM, 78 mM, 80 mM, 85 mM, 88 mM, 90 mM, 95 mM, 98 mM, 100 mM, or any range between these point values.

[0024] In some embodiments, preferably, the stabilizer is selected from one or more of sucrose, trehalose, sorbitol, mannitol, proline or a pharmaceutically acceptable salt, methionine or a pharmaceutically acceptable salt, phenylalanine or a pharmaceutically acceptable salt, leucine or a pharmaceutically acceptable salt, glycine or a pharmaceutically acceptable salt, valine or a pharmaceutically acceptable salt.

[0025] In some embodiments, the solubilizing agent is selected from polysorbate 20, polysorbate 80, polyoxyl, Triton, sodium lauryl sulfate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl- sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl- sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristelamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristelamidopropyl-betaine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl taurate, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymer of ethylene and propylene glycol, and the like; in some embodiments, the solubilizing agent is selected from polysorbate 20 or polysorbate 80.

[0026] In some embodiments, the solubilizing agent is present at a concentration of about 0.1 mg / mL to about 3.0 mg / mL; in some embodiments, the solubilizing agent is present at a concentration of about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.3 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, 2.3 mg / mL, 2.5 mg / mL, 2.8 mg / mL, 3 mg / mL, or any range between these point values.

[0027] In some embodiments, the pharmaceutical composition further comprises a buffer; in some embodiments, the buffer is selected from a histidine salt buffer, a succinate buffer, a histidine-aspartate salt buffer; in some embodiments, the histidine salt buffer is selected from a histidine-acetic acid buffer or a histidine-hydrochloric acid buffer; the succinate buffer is selected from a succinic acid-sodium succinate buffer.

[0028] In some embodiments, the concentration of the buffer is about 0 to about 20 mM; in some embodiments, the concentration of the buffer is about 0 mM, 1 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, 13 mM, 13.5 mM, 14 mM, 14.5 mM, 15 mM, 15.5 mM, 16 mM, 16.5 mM, 17 mM, 17.5 mM, 18 mM, 18.5 mM, 19 mM, 19.5 mM, 20 mM, or any range between these point values.

[0029] In some embodiments, the pH of the pharmaceutical composition is about 4.0 to about 6.0; in some embodiments, the pH of the pharmaceutical composition is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, or any range between these point values.

[0030] The final pH of the pharmaceutical composition in the present disclosure is almost consistent with the buffer pH. But it is well known to those skilled in the art that there can be pH drift during the preparation of pharmaceutical formulations, and the drift of the final pH of the pharmaceutical formulation in the present disclosure is within ±0.3.

[0031] In some embodiments, the pharmaceutical composition of the present disclosure comprises the following components: about 1 mg / mL to about 100 mg / mL of an anti-GPC3 and CD3 bispecific antigen binding molecule, (b) about 20 mg / mL to about 100 mg / mL of a sugar and / or about 0-100 mM of an amino acid, (c) about 0.1 mg / mL to about 3.0 mg / mL of polysorbate 80, and (d) about 0 to about 20 mM of succinate buffer; the pH of the pharmaceutical composition is about 4.0 to about 6.0.

[0032] In some embodiments, the present disclosure provides a lyophilized formulation which can form the pharmaceutical composition described in the present disclosure after reconstitution.

[0033] In some embodiments, the present disclosure provides a lyophilized formulation which is obtained by freeze-drying the pharmaceutical composition described in the present disclosure.

[0034] In some embodiments, the present disclosure provides a method for preparing a lyophilized formulation, which comprises the step of freeze-drying the pharmaceutical composition described in the present disclosure. In some embodiments, the present disclosure provides a method for preparing a pharmaceutical composition, which comprises the step of mixing the following components: (a) about 1 mg / mL to about 100 mg / mL of an anti-GPC3 and CD3 bispecific antigen binding molecule, (b) about 20 mg / mL to about 100 mg / mL of a sugar and / or about 0-100 mM of an amino acid, (c) about 0.1 mg / mL to about 3.0 mg / mL of polysorbate 80, and (d) about 0 to about 20 mM of succinate buffer; the pH of the pharmaceutical composition is about 4.0 to about 6.0.

[0035] In some embodiments, the present disclosure provides use of the pharmaceutical composition, the lyophilized formulation of the present disclosure in the preparation of a medicament for treating or preventing a disease associated with GPC3; preferably, the disease is cancer; more preferably, the cancer is selected from hepatocellular carcinoma, gastric cancer, esophageal cancer, ovarian cancer or non-squamous non-small cell lung cancer.

[0036] In some embodiments, the present disclosure provides a method of treating or preventing a disease associated with GPC3; the method comprises administering to a subject the pharmaceutical composition, the lyophilized formulation of the present disclosure; preferably, the disease is cancer; more preferably, the cancer is selected from hepatocellular carcinoma, gastric cancer, esophageal cancer, ovarian cancer or non-squamous non-small cell lung cancer. DETAILED DESCRIPTION

[0037] All publications, patents, and patent applications mentioned in the present disclosure are incorporated by reference herein to the extent such reference does not conflict with express statements made herein.

[0038] Before the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0039] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure can be described in terms of ranges of values. Unless otherwise stated, it is to be understood that numerical ranges or ranges described in terms of ranges are for the purpose of description only and are not intended to limit the scope of the present disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible individual numerical values within that range, as if each and every such subrange and numerical value were specifically written herein. The same principles apply to ranges recited using endpoints based on approximations. Such principles apply to ranges recited using endpoints based on approximations. Such principles apply regardless of the breadth of the range or the approximation.

[0040] Terminology

[0041] The term "about" includes and describes the value or parameter itself. For example, "about x" includes and describes "x" itself. As used herein, the term "about," when used in conjunction with a measured value or in reference to a value, unit, constant, or series of values, means a variance of ± 1-10%. In some embodiments, the term "about," when used in conjunction with a measured value or in reference to a value, unit, constant, or series of values, means a variance of ± 1%, ± 2%, ± 3%, ± 4%, ± 5%, ± 6%, ± 7%, ± 8%, ± 9%, or ± 10%.

[0042] The three letter code and one letter code for amino acids used herein are as described in J. Biol. Chem, 243, p 3558 (1968).

[0043] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0044] The term "antigen binding molecule" as used herein refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and derivatives thereof, e.g., fragments.

[0045] The term "bispecific" means that the antigen binding molecule is capable of specifically binding to two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of the two antigen binding sites being specific for a different antigenic determinant. In certain embodiments, the bispecific antigen binding molecule is capable of binding to two antigenic determinants simultaneously, especially two antigenic determinants expressed on two different cells.

[0046] The term "antigen" refers to a substance that is recognized and bound specifically by an antibody or antigen-binding fragment. Broadly, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, a polyepitope, a single domain, a multi-domain, or an entire extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof can all constitute an antigen. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, among others. An "antigen" can also refer to a molecule that elicits an immune response. Any form of an antigen or a cell or preparation containing the antigen can be used to generate antibodies specific for an antigenic determinant. An antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with a cell expressing at least a portion of the antigen on its surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein) or a protein construct (e.g., Fc antigen). The antigen can be produced in a genetically modified cell. Any of the foregoing antigens can be used alone or in combination with one or more immunogenicity-enhancing adjuvants known in the art. DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of an ECD sufficient to elicit an immunogenic response. Any vector can be used to transform the cell in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0047] The term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both by contiguous amino acids or noncontiguous amino acids juxtaposed by an protein's tertiary structure. Epitopes formed from contiguous amino acids are typically preserved when the protein is denatured, whereas epitopes formed by tertiary structure are typically lost upon denaturation and may, therefore, be exposed or concealed depending on an protein's conformational state. Epitopes are usually conformational in nature, and include at least 3-15 amino acids. Methods of determining an epitope to which a given antibody binds are well known in the art, including immunoblot and immunoprecipitation detection assays, among others. Methods of determining the spatial conformation of an epitope include techniques in the art and described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

[0048] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear, cyclic, or branched, they can comprise modified amino acids, particularly conservative modifications of amino acids, and they can be interrupted by non-amino acids. The terms also encompass modified amino acid polymers, for example, by sulphation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation with a labeling component, etc. As used herein, the term "amino acid" refers to both natural and / or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the origin of the polypeptide. The term also encompasses polypeptides expressed from the specified nucleic acid sequence.

[0049] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions and / or deletions in a polypeptide sequence. An "amino acid substitution" or "substitution" or "replacement" herein means the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, the substitution S32A means that the serine at position 32 is replaced with alanine.

[0050] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0051] Conventional immunoglobulins are tetramers consisting of two heavy and two light chains, combining a molecular weight of about 150 kDa. In Camelidae members, a considerable proportion of serum antibodies are homodimeric IgG with a molecular weight of about 80 kD (Hamers-Casterman et al., 1993, Nature, 363, 446-448). These heavy-chain immunoglobulins (Ig) comprise three domains, the variable domain of heavy-chain antibody (VHH) of which is the variable region. Recombinant VHHs (about 12 to 14 kD) constitute complete antigen-binding domains and show a broad antigen-binding spectrum. Their hypervariable regions are enlarged and exhibit unique characteristics, such as three to four hydrophobic framework residues (which interact with VL of conventional antibodies) are replaced by more hydrophilic amino acids. To stabilize the enlarged CDRs, in addition to the conventional disulfide bond, VHHs can have an additional disulfide bond between CDR1 and CDR3 in single-humped camels, and between CDR2 and CDR3 in llamas (Harmsen and De Haard, 2007, Appl Microbiol Biotechnol., 77, 13-22; Muyldermans, 2001, J Biotechnol., 74, 277-302). The enlarged CDR3 loop can adopt a convex conformation, whereas the conventional paratope is restricted to a concave or planar structure (Muyldermans, 2001, J Biotechnol., 74, 277-302). These features allow VHHs to recognize unique epitopes that are less immunogenic for conventional antibodies (Lafaye, 2009, Mol Immuno., 46, 695-704; Wernery, 2001, J Vet Med B Infect Dis Vet Public Health., 48, 561-568). Although VHHs are defined as monovalent antibodies, defaulting to exclude any avidity effects, the biological activity measured as in vitro IC50 can be similar to that of conventional bivalent antibody molecules (Thys et al., 2010, Antiviral Res., 87, 257-264).

[0052] The term antibody "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. In the present context, the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) each comprise four conserved framework regions (FRs) and three complementarity determining regions (CDRs). Within the term "complementarity determining region" or "CDR" refers to a region within a variable domain that is primarily responsible for binding an antigen; the "framework" or "FR" refers to the variable domain residues other than the CDR residues. The VHand VLeach comprise three CDR regions: HCDR1, HCDR2, and HCDR3; LCDR1, LCDR2, and LCDR3. Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus (also N-terminus) to carboxy-terminus (also C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0053] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM, with the heavy chain constant domains designated a, d, e, g, and m, respectively. IgG and IgA can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0054] In the case of IgG, IgA, and IgD antibodies, the constant region comprises three domains, termed CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible "hinge" region, which is a variable length segment rich in proline and cysteine. Each class of antibody further comprises inter- and intra-chain disulfide bonds formed between paired cysteine residues.

[0055] It is noted that the CDRs and FRs of the antibody variable regions herein are determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Thus, humanized antibodies comprising one or more CDRs derived from any of the naming systems, based on the antibody sequences herein, are expressly maintained within the scope herein.

[0056] The term "GPC3", also known as Glypican 3 or Glypican 3, as used herein, is a type of proteoglycan attached to the cell surface by a glycerophosphatidylinositol (GPI) anchor, belonging to the heparan sulfate proteoglycan (HSPG) family, mainly involved in the regulation of cell proliferation, adhesion and migration and other processes. GPC3 gene is located on human X chromosome (Xq26.1), which encodes a GPC3 protein precursor containing 580 amino acid residues (Nat Genet, 1996, 12(3): 241-7.). The GPC3 core protein is about 70 KDa, rich in a unique sequence containing 14 cysteine residues (Cys), and furin protease produces about 40 KDa soluble N-terminal peptide and about 30 KDa membrane-bound C-terminal peptide containing 2 heparan sulfate (HS) sugar chains by cleaving the core protein (between Arg358 and Cys359) (Eur J Cancer, 2011, 47(3): 333-8.; Proc Natl Acad Sci USA, 2011, 108(32): 13112-7.).

[0057] The term "anti-GPC3 / CD3 antibody" as used herein refers to an antibody that can bind to GPC3 and CD3. Exemplary GPC3 / CD3 antibodies or antigen-binding fragments thereof are described in the Examples of the present disclosure, which are also described in the patent application filed by the applicant of the present application (PCT patent application number: PCT / CN2023 / 128662, entitled: A bispecific antibody against glypican 3 and its application, the entire contents of which are incorporated herein by reference.

[0058] The term "sequence identity" or "sequence similarity" or "sequence homology" refers to the percentage of amino acid residues in a candidate sequence that are identical with the same amino acid residues in a reference polypeptide sequence, after aligning the sequences (and introducing gaps, if necessary) to achieve the maximum percentage sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage of amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. A person skilled in the art can determine the appropriate parameters for alignment, including any algorithm required to obtain the maximum alignment over the entire length of the sequences compared.

[0059] In certain embodiments, reference can be made herein to chimeric camelid / human antibodies, particularly chimeric antibodies in which the VH and / or VL domains are entirely of camelid sequence (e.g., llama or alpaca) while the remainder of the antibody is entirely of human sequence. Also included in some preferred embodiments herein are "humanized" or "germlined" camelid antibodies as well as camelid / human chimeric antibodies in which the VH and / or VL domains contain one or more amino acid substitutions in the framework regions relative to camelid VH and / or VL domains obtained by active immunization. Such "humanization" processes increase the percentage of sequence identity with human germline VH or VL domains by substituting non-matching amino acid residues in the starting camelid VH or VL domain with the corresponding residues in a human germline VH or VL domain.

[0060] Included herein are native, recombinant VHH or VH.

[0061] A VHH according to herein can be in monomeric form or in a homo-multimeric form, such as a homo-dimer or a homo-trimer.

[0062] An antibody according to herein includes a camelid-derived antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.

[0063] The term "chimeric antibody" is a construct in which a portion of the heavy and / or light chain is identical with, or homologous to, a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with, or homologous to, a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and / or a fragment of such antibody. In a narrow sense, a chimeric antibody comprises all or most of a selected murine heavy and light chain variable region operably linked to human light and heavy chain constant regions. Constant region sequences, or variants or derivatives thereof, can be operably associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide a full-length anti-GPC3 antibody that can be used per se or can be incorporated into an antibody according to herein.

[0064] The term "humanized antibody" is a hybrid immunoglobulin, immunoglobulin chain or fragment thereof, which contains minimal sequence from a non-human immunoglobulin. In most instances, a humanized antibody will be a human immunoglobulin (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or primate that have the desired specificity, affinity, and / or capacity. In some instances, framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In certain instances, "backmutating" can be introduced into a humanized antibody, in which a residue or residues in the variable region of the recipient human antibody are replaced by the corresponding residue from the donor non-human species antibody. Such backmutations can help to maintain the proper three-dimensional conformation of the grafted CDR(s) and thus improve affinity and antibody stability. Antibodies from a variety of donor species can be used, including but not limited to mouse, rat, rabbit, or non-human primate. Additionally, a humanized antibody can contain new residues not found in the recipient antibody or in the donor antibody, to further improve antibody performance.

[0065] The term "single domain antibody" also known as nanobody, can be defined as an amino acid sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Wherein FR1-FR4 refer to the Frame regions 1-4, respectively, and wherein CDR1-CDR3 refer to the Complementarity Determining Regions 1-3, respectively. "VHH" relates to the variable antigen binding domain from heavy chain antibodies of Camelidae (Camelus dromedarius, Camelus bactrianus, Lama glama, Lama pacos, etc.) (see Nguyen, 2000 EMBO J., 19, 921-930; Muyldermans, 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot, 2011, Antiviral Res., 92, 389-407).

[0066] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (e.g. via a synthetic linker such as a short flexible polypeptide linker) and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, the scFv can have the VL and VH variable regions in any order (e.g. with respect to the N-terminus and C-terminus of the polypeptide), the scFv can comprise VL-peptide linker-VH or can comprise VH-peptide linker-VL.

[0067] The term "Fc" is used herein to define a C-terminal region of an immunoglobulin heavy chain, i.e., a dimeric polypeptide chain comprising the C-terminal constant domains that associate to form a stable unit in an immunoglobulin heavy chain. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to stretch from Cys226, or Pro230, to the carboxy-terminus of the heavy chain, for example, the IgG Fc domain comprises IgG CH2 and IgG CH3 constant domains. Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0068] "Pharmaceutical composition" means a mixture of one or more of the antibodies described herein with other chemical components, such as a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of an active ingredient to a subject and to facilitate absorption of the active ingredient.

[0069] The terms "formulation" or "pharmaceutical formulation" or "prescription" or "pharmaceutical prescription" are used interchangeably herein to mean a form that allows the active pharmaceutical ingredient to be present effectively, and which does not contain other components that are toxic to the subject to which the formulation is to be administered.

[0070] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0071] In the present disclosure, the solvent in the solution form of the pharmaceutical composition is water, unless otherwise specified.

[0072] The term "pharmaceutically acceptable excipient" is a substance / agent that can be reasonably administered to a subject to provide an active pharmaceutical ingredient in a stable formulation form in an effective amount. Suitable pharmaceutically acceptable excipients are well known in the art and include, but are not limited to, buffers, stabilizers, solubilizers, and the like.

[0073] The term "buffer" refers to an agent that maintains the pH of a solution of a formulation within an acceptable range. Examples of buffers that control the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0074] The term "stabilizer" is a pharmaceutically acceptable excipient used to protect the active pharmaceutical ingredient and / or the formulation from chemical and / or physical degradation during production, storage and application.

[0075] The term "solubilizer" means a pharmaceutically acceptable excipient used to protect the protein formulation from mechanical stress such as agitation and shear.

[0076] The term "lyophilized formulation" refers to a composition obtained or obtainable by a freeze-drying process of a liquid formulation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0077] The pharmaceutical composition according to the present disclosure is capable of achieving a stable effect: a pharmaceutical composition wherein the antibody substantially retains its physical stability and / or chemical stability and / or biological activity after storage, preferably, a pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. There are various analytical techniques to measure the stability of a protein after storage for a selected period of time at a selected temperature.

[0078] The term "treatment" refers to clinical intervention of an individual or cells with the intent to alter the natural course of a disease, whether to prevent or delay a clinical pathologic process. Therapeutic effects include, but are not limited to, preventing or delaying the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, ameliorating or palliating the disease state, relieving or improving the prognosis, etc.

[0079] The term "individual" or "subject" or "patient" refers to any animal, such as a mammal or a marsupial. The individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus monkey or rhesus monkey or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any kind of poultry.

[0080] The term "disease" or "disorder" or "condition" or the like refers to any alteration or disturbance in the normal functioning of cells, tissues, or organs that causes harm or interference. For example, the "disease" includes, but is not limited to, a tumor, a pathogen infection, an autoimmune disease, a T cell dysfunction disease, or a deficiency in the ability to immune tolerance (such as transplant rejection), etc.

[0081] The term "tumor" refers to a disease characterized by pathological proliferation of cells or tissues, and subsequent migration or invasion of other tissues or organs. Tumor growth is generally uncontrolled and progressive, without inducing or suppressing normal cell proliferation. Tumor includes "cancer", which refers to all malignant tumors.

[0082] Examples

[0083] The present disclosure is described in detail below by way of examples. It is necessary to point out that the following examples are merely for further illustration of the present disclosure and are not to be construed as limiting the scope of protection of the present disclosure. Non-essential improvements and adjustments made by those skilled in the art based on the above-described invention remain within the scope of protection of the present disclosure. Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments used is not specified, they are all commercially available conventional products.

[0084] The following methods are used to carry out relevant determinations in the examples:

[0085] Protein content determination: The protein content of this product was determined using a Lunatic microspectrophotometer (Unchained Labs, Little Lunatic). The extinction coefficient of this product is 1.75. The protein content (mg / ml) is determined by subtracting the absorbance at 330 nm from the absorbance at 280 nm and dividing the result by the extinction coefficient.

[0086] Size Exclusion Chromatography (SEC-HPLC): Size exclusion chromatography was used to determine the size heterogeneity of this product. The column used in this method was Waters BioResolve TM SEC mAb Column( 2.5 μm, 7.8 × 300 mm) and run on a Waters e2695-2489 HPLC system; detection wavelength was 280 nm; the mobile phase consisted of 100 mM sodium phosphate, 250 mM sodium chloride buffer, pH 6.8 ± 0.1; the flow rate was 0.5 ml / min, and the isocratic run was 30 min. The sample was centrifuged, the supernatant was transferred to an injection vial, and 50 μg was injected. Empower 3 software (Waters) was used for integration, and the peak area percentage (content) of each peak was calculated by area normalization. HMWS is the sum of the contents of each high molecular weight species, and LMWS is the sum of the contents of each low molecular weight species.

[0087] Isoelectric focusing capillary electrophoresis (iCIEF): Imaging capillary isoelectric focusing electrophoresis is used to determine the charge heterogeneity of the product. In this method, the capillary is a FC-coated fused quartz capillary with an inner diameter of 100 pm, and the effective separation length is 5 cm; 10% glycerol, 4 M urea, 4% Cytiva pharmalyte 3-10, and 10 mM arginine solution are added to the sample at a final concentration of 0.20 mg / ml during sample processing; the focusing separation voltage and time are 1.5 kV-1 min and 3 kV-13 min, respectively. The Empower 3 instrument control software is used for direct analysis or the data is exported after data acquisition software, and then Empower 3 software (Waters Corporation) is used for integral processing. The peak area percentage (content) of each peak is calculated by area normalization method. The acid region content is the sum of the contents of each acidic variant, and the base region content is the sum of the contents of each basic variant.

[0088] Reduced capillary electrophoresis (rCE-SDS): Reduced capillary electrophoresis is used to determine the rCE-SDS purity of the product. In this method, the capillary is a non-coated fused quartz capillary with an inner diameter of 50 pm, and the effective separation length is 20 cm (total length 30.2 cm); a PDA detector is used, the wavelength is 220 nm, the bandwidth is 10 nm, the Channel Data Rate is 4 Hz, the window number is 8; the sample chamber temperature is set to 25°C, and the capillary temperature is set to 25°C; the sample processing solution is 20 mM disodium hydrogen phosphate-citric acid, 1% SDS, pH 6.0. During sample processing, 100 pg of test product protein is added to 5 pl of beta-mercaptoethanol, heated at 70°C for 10 min, injected at 5 kV for 20 s, separated at 15 kV for 30 min, and the corrected peak area percentage (content) of each peak is calculated by area normalization method. The sum of the contents of chain 1 (C1) and chain 2 (C2) is the product purity, HMWS is the sum of the contents of each high molecular weight substance, and LMWS is the sum of the contents of each low molecular weight substance.

[0089] Non-reduced capillary electrophoresis (nrCE-SDS): nrCE-SDS purity of the product is determined by non-reduced capillary electrophoresis. In this method, the capillary is an uncoated fused silica capillary with an inner diameter of 50 μm, the effective separation length is 20 cm (total length 30.2 cm); PDA detector is used, wavelength 220 nm, bandwidth 10 nm; Channel Data Rate: 4 Hz; Window 8; The sample chamber is set to 25 °C, and the capillary is set to 25 °C; The sample treatment solution is 20 mM disodium hydrogen phosphate-citric acid, 1% SDS, pH 6.0. During sample processing, 100 μg of test product protein is added to NEM with a final concentration of 5 mM, heated at 70 °C for 10 min, injected at 5 kV-20 s, separated at 15 kV-35 min, and the corrected peak area percentage (content) of each peak is calculated by area normalization method. HMWS is the sum of the contents of each high molecular weight substance, and LMWS is the sum of the contents of each low molecular weight substance.

[0090] Binding activity: the binding ability of the product to GPC3 and CD3 proteins is determined by ELISA method. First, human GPC3 protein is coated on a 96-well plate, then a gradient-diluted test product is added, and then a fixed-concentration biotin-labeled CD3 (Biotin-CD3) protein is added. One end of the test product binds to the GPC3 protein, and the other end binds to the biotin-labeled CD3 protein. Streptavidin labeled with horseradish peroxidase is added to the 96-well plate, and finally TMB substrate solution is added for color development, and the absorbance value at 450 nm is read. Using the four-parameter fitting regression model, the SoftMax Pro computer software is used to draw the curve, and by comparing the EC50 values of the reference and test products, the relative binding activity of the test product to CD3 and GPC3 proteins is calculated. The calculation formula is:

[0091] Micro flow imaging analysis (MFI): the particulate matter of the test product is characterized by micro flow imaging analysis technology. The micro flow imaging particle analysis system integrates digital microscopy, microfluidics and image processing technology into a precision instrument for automatically analyzing particulate matter. When the sample flows through the flow cell (detection area), the MFI system captures the particulate matter in the sample, and counts the particulate matter by software. In this method, the sample volume is 1.0 ml, the total effective volume is 0.7 ml, the analysis volume is 0.51 ml, the MVSS software (Protein Simple company) is used for analysis, and the number of microparticles ≥2 μm, 5 μm, 10 μm, 25 μm, 50 μm per milliliter of test product is recorded, and the main morphological classification of microparticles ≥5 μm is performed.

[0092] The anti-GPC3 / CD3 antibody or antigen-binding fragment thereof described in the present disclosure is a bispecific antibody targeting GPC3 and CD3 simultaneously, the preparation and screening of which are described in detail in PCT / CN2023 / 128662, which is incorporated herein in its entirety. Exemplarily, the anti-GPC3 / CD3 antibody used in the following formulation examples is as follows: the complete molecule of the antibody is composed of two different chains (first peptide chain (Knob chain) and second peptide chain (Hole chain), respectively). The N-terminal of the first peptide chain (C1) is a single-domain antibody heavy chain variable region (VHH, Variable Domain of Heavy Chain Antibody) targeting GPC3, and the C-terminal thereof is connected with the Fc segment of IgG4. The N-terminal of the second peptide chain (C2) is a CD3 single-chain antibody (Single-chain variable fragment, scFv) targeting CD3, and the C-terminal thereof is connected with the Fc segment of IgG4. The sequence of the first peptide chain is shown in SEQ ID NO: 12, wherein the sequence of the heavy chain variable region of the first peptide chain is shown in SEQ ID NO: 10; the sequence of the second peptide chain is shown in SEQ ID NO: 13, wherein the sequence of the scFv of the second peptide chain is shown in SEQ ID NO: 11.

[0093] Example 1: Solubilizer type and concentration test

[0094] Test examples A1-A6 are formulations containing different solubilizer types and concentrations. Test examples A1-A3 contain different concentrations of polysorbate 20, and test examples A4-A6 contain different concentrations of polysorbate 80. The content of each component is shown in Table 1.

[0095] Table 1 Formulation prescription containing different solubilizer types and concentrations

[0096] The stability of each prescription under the following different conditions was tested respectively:

[0097] The prepared samples were subjected to the influence factor test. Under high temperature (40℃±2℃) conditions, sampling detection was performed at 0 day, 7 days, 14 days, and 28 days, respectively; under accelerated (25℃±2℃, relative humidity 60%±5%) conditions, sampling detection was performed at 30 days; under oscillation (25℃±2℃, 150 rpm) conditions, sampling detection was performed at 5 days.

[0098] The results of the high-temperature storage stability experiment are shown in Table 2, the results of the accelerated storage stability experiment are shown in Table 3, and the results of the oscillation storage stability experiment are shown in Table 4.

[0099] Table 2 High-temperature storage stability results of anti-GPC3 / CD3 antibody formulations

[0100] Note: ND not detected; N / A not tested.

[0101] Table 3 Accelerated storage stability results for anti-GPC3 / CD3 antibody formulations

[0102] Note: ND not detected; N / A not tested.

[0103] Table 4 Oscillatory storage stability results for anti-GPC3 / CD3 antibody formulations

[0104] Note: ND not detected; N / A not tested.

[0105] Conclusion: After 28 days at high temperature (40°C ± 2°C), 30 days at accelerated (25°C ± 2°C), and 5 days at oscillation (25°C ± 2°C, 150 rpm), the sample A1-A6 showed consistent trends in the changes of the test indicators, and no significant differences were observed, indicating that the formulations in each group had good stability.

[0106] Example 2: Screening of buffer system types

[0107] The types of buffer systems of the formulations were studied, and the prescription design is shown in Table 5. To prepare the formulation prescription solution, different types of buffer systems were used, and 30 kDa ultrafiltration centrifuge tubes were used for ultrafiltration and replacement, and aspartic acid was used to adjust the pH value in the test example with 0 mM buffer. The prepared formulation was sterile filtered through a 0.22 μm low protein binding filter, and was filled into sterile tube bottles under sterile conditions, and was sealed with a film-coated rubber stopper and an aluminum plastic combined cover.

[0108] Table 5 Prescription design for screening of different buffer system types

[0109] The stability of each prescription under high temperature storage conditions (placed at 40°C ± 2°C for 14 days) and light conditions (25°C, 5000 lx, 5 days) was tested respectively, and the high temperature

[0110] The results of the high temperature (40°C ± 2°C) and light storage stability experiments are shown in Table 6.

[0111] Table 6 High temperature (40°C ± 2°C) and light storage stability results for screening of different buffer systems

[0112] Note: ND not detected; N / A not tested.

[0113] The experimental results show that:

[0114] After 14 days of storage at high temperature (40°C ± 2°C), the nrCE-SDS monomer purity of samples B1-B4 decreased by at most 2.3%, and the iCIEF purity decreased in a basically consistent manner; after 5 days of storage under light (25°C, 5000lx), the nrCE-SDS monomer purity decreased by at most 1.7%, and the iCIEF purity decreased in a basically consistent manner. The results show that the formulations of each group have good stability.

[0115] Example 3: Buffer system concentration screening test

[0116] The effect of the buffer system concentration of the formulation on the stability of the protein was studied. The prescription design is shown in Table 7. To prepare the formulation prescription solution, a buffer system containing different concentrations of sodium succinate was used, and a 30 kDa ultrafiltration centrifuge tube was used for ultrafiltration and replacement. In the test example with 0 mM buffer, aspartic acid was used to adjust the pH value. The prepared formulation was sterile filtered through a 0.22 μm low protein binding filter, and was filled into sterile tube bottles under sterile conditions, sealed with a film-coated rubber stopper and an aluminum plastic combined cover.

[0117] Table 7: Prescription design for screening of different concentrations of buffer system

[0118] The stability of each prescription under high temperature storage conditions (storage at 40°C ± 2°C for 14 days) was tested, and the results of the high temperature storage stability experiment are shown in Table 8.

[0119] Table 8: High temperature (40°C ± 2°C) storage stability results of different concentrations of buffer system screening

[0120] Note: ND, not detected; N / A, not tested.

[0121] The experimental results show that:

[0122] After 14 days of storage at high temperature (40°C ± 2°C), the SEC-HPLC monomer purity of samples C1-C4 decreased by at most 3.4%. The results show that the formulations with a buffer concentration in the range of 0-20 mM have good stability.

[0123] Example 4: Stabilizer type screening test

[0124] The effects of various types of stabilizers (e.g., sucrose, trehalose, sorbitol, mannitol) on protein stability were studied. The formulation designs are shown in Tables 9 and 10. Different types of stabilizers were used. The formulated preparations were sterile filtered through a 0.22 μm low protein binding filter and aseptically filled into sterile vials. E1, E2, F1, F2, and F3 were stored under injection solution conditions using a laminated stopper and aluminum cap.

[0125] Table 9 Formulation designs for screening of different stabilizer types

[0126] Table 10 Formulation designs for screening of different stabilizer types

[0127] Each formulation was tested for stability under high temperature storage conditions (40°C ± 2°C for 14 days), with a focus on monitoring SEC-HPLC purity and nrCE-SDS purity of the samples after high temperature storage. The results of the high temperature storage stability test are shown in Tables 11 and 12.

[0128] Table 11 Results of high temperature (40°C ± 2°C) storage stability for screening of different stabilizer types

[0129] Note: ND, not detected; N / A, not tested. Table 12 Results of high temperature (40°C ± 2°C) storage stability for screening of different stabilizer types

[0130] Note: ND, not detected; N / A, not tested.

[0131] The experimental results showed that:

[0132] After 14 days of storage at high temperature (40°C ± 2°C), the SEC-HPLC monomer purity of samples E1-E2 decreased by at most 2.4%, and the nrCE-SDS purity of the two samples showed a similar trend. The SEC-HPLC purity and change trend of F1-F3 were basically consistent. This result showed that the formulations with stabilizers selected from sucrose, trehalose, sorbitol, and mannitol all had good stability.

[0133] Example 5 Screening of Amino Acid Content and Types

[0134] The effects of various amino acids (e.g., proline, methionine, phenylalanine, valine, ornithine) and combinations of amino acids (e.g., methionine, proline, leucine) on the stability of the formulations were studied. The prescription designs are shown in Tables 13 and 14. Different types of amino acids were added to the solution. The formulated formulations were sterile filtered through a 0.22 μm low protein binding filter and filled into sterile vials under aseptic conditions, and stored using a film-coated stopper and an aluminum cap.

[0135] Table 13 Prescription design for screening of different amino acid types

[0136] Table 14 Prescription design for screening of different combinations of amino acid types

[0137] The stability of each formulation was tested under high-temperature storage conditions (placed at 40°C ± 2°C for 14 days) and light conditions (25°C, 5000 lx, 10 days), respectively. The SEC detection was performed in two ways: original concentration of protein content and diluted to 5 mg / ml. The results of the high-temperature storage stability experiment are shown in Tables 15, 16, and 17.

[0138] Table 15 High-temperature (40°C ± 2°C) storage stability results of different amino acid types

[0139] Note: SEC-HPLC purity was detected at the original concentration of protein content.

[0140] Table 16 High-temperature (40°C ± 2°C) and light (25°C, 5000 lx, 10 days) storage stability results of screening of different amino acid types

[0141] Note: SEC-HPLC purity was detected at a protein concentration of 5 mg / ml; ND, not detected.

[0142] Table 17 High-temperature (40°C ± 2°C) and light (25°C, 5000 lx, 5 days) storage stability results of different combinations of amino acid types

[0143] Note: SEC-HPLC purity was detected at the original concentration of protein content; ND, not detected.

[0144] The experimental results showed that:

[0145] According to the results in Table 15, the initial SEC-HPLC monomer purity of the sample is significantly reduced after adding ornithine in the solution; according to the results in Table 16, the SEC-HPLC purity of the sample decreases in a basically consistent trend after adding proline, methionine, phenylalanine, valine in the solution and placing under high temperature for 14 days or under light conditions for 10 days; according to the results in Table 17, the SEC-HPLC purity of the sample decreases in a basically consistent trend after adding the amino acid combination of proline, methionine, leucine, etc. in the solution. The results show that the preparations of proline, methionine, phenylalanine, valine, leucine and their combinations in the solution all have good stability.

[0146] Example 6 Screening test of various auxiliary material content range

[0147] The protein concentration, the concentration of stabilizer, the concentration of solubilizer and the pH range were screened to investigate the influence on the stability of the protein. The prescription design is shown in Table 18. The stabilizers are taken as sucrose, proline and glycine for example, and the solubilizers are taken as polysorbate 20 and polysorbate 80 for example. The prepared preparation is sterilized by a 0.22 μm low protein binding filter, and is filled into a sterile tube under sterile conditions. The injection freeze-dried preparation is obtained after freeze-drying by a freeze-drying machine, and is stored after adding a rubber plug and an aluminum cap.

[0148] Table 18 Prescription design of screening test of various auxiliary material content range

[0149] The stability of each prescription under high temperature storage condition (placed at 40℃±2℃ for 14 days) was tested respectively. The high temperature storage stability test results are shown in Table 19.

[0150] Table 19 High temperature (40℃±2℃) and light (25℃, 5000lx, 5 days) storage stability results of screening of different amino acid types

[0151] Note: The SEC-HPLC purity was detected under the condition of 5 mg / ml protein concentration, and ND means not detected.

[0152] The 0-day sample of R1, R3 and R4 prescriptions was detected for soluble microparticles (method: MFI) to evaluate the influence of different concentrations of polysorbate content on the insoluble microparticles of the product, and the results are shown in Table 20.

[0153] Table 20 Insoluble microparticle detection results of screening of different amino acid types

[0154] The experimental results found that:

[0155] According to the results in Table 19, when the protein concentration is 1-100 mg / ml, the sugar concentration is 20-100 mg / ml, the amino acid concentration is 10-100 mg / ml, the solubilizer concentration is 0.1-3 mg / ml, and the pH value is in the range of 4.0-6.0, the stability change trends of the samples under the conditions of high temperature (40°C±2°C) for 14 days and light (25°C, 5000 lx) for 5 days are basically the same, and all meet the quality standards of the product. According to the results in Table 20, the concentration of the solubilizer does not affect the number of insoluble particles in the product, which is within the acceptable range. At the same time, according to the change trend of the prescription of R3, it is shown that the preparation added with glycine also has good stability.

[0156] Example 7 Long-term, accelerated stability of the preparation containing the anti-GPC3 / CD3 antibody

[0157] The following preparation prescription was selected: 5 mg / ml anti-GPC3 / CD3 antibody, 90 mg / ml sucrose, 0.5 mg / ml polysorbate 20, 5 mM sodium succinate buffer, and pH value of 4.2-4.8. Stability studies were conducted on the above selected preparation, including: forced condition test (high temperature test), accelerated test, long-term test. The sample placement mode in the stability study was upright; the long-term, accelerated, and high-temperature samples were all packaged in a simulated marketing package. The investigation conditions are shown in Table 21:

[0158] Table 21 Investigation conditions for stability experiment of the preparation containing the anti-GPC3 / CD3 antibody

[0159] The stability results of the preparation under the above investigation conditions are shown in Table 22:

[0160] Table 22 Long-term test (upright) results of the preparation containing the anti-GPC3 / CD3 antibody (2-8°C)

[0161] Note: ND, not detected.

[0162] From the above results, it can be seen that at 24 months, the comprehensive activity of the preparation reached 95%, indicating that the preparation prescription of the present disclosure has good stability.

[0163] Table 23 Accelerated test (upright) results of the preparation containing the anti-GPC3 / CD3 antibody (25±2°C)

[0164] Note: ND, not detected.

[0165] From the above results, it can be seen that at 6 months, the comprehensive activity of the preparation reached 108%, indicating that the preparation prescription of the present disclosure has good stability.

[0166] Table 24 contains the results of the high temperature test (upright) of the anti-GPC3 / CD3 antibody formulation (40°C ± 2°C)

[0167] Note: ND, not detected.

[0168] From the above results, it can be seen that the overall activity of the formulation reached 99% at 30 days, indicating that the formulation of the present disclosure has good stability.

Claims

1. A pharmaceutical composition comprising an anti-GPC3 / CD3 bispecific antigen binding molecule and an excipient, wherein the anti-GPC3 / CD3 bispecific antigen-binding molecule comprises a first antigen-binding domain that recognizes glypican 3 (GPC3), and a second antigen-binding domain that recognizes the CD3 subunit of the T cell receptor; The first antigen-binding domain comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; The second antigen binding domain comprises an scFv, wherein the scFv comprises a HCDR1 as shown in SEQ ID NO: 4, a HCDR2 as shown in SEQ ID NO: 5, and a HCDR3 as shown in SEQ ID NO: 6, and a LCDR1 as shown in SEQ ID NO: 7, a LCDR2 as shown in SEQ ID NO: 8, and a LCDR3 as shown in SEQ ID NO: 9; The auxiliary material is optionally selected from one or more of a stabilizer and a solubilizer; Preferably, the sequence of the heavy chain variable region of the first antigen-binding domain is shown in SEQ ID NO: 10, and the sequence of the scFv of the second antigen-binding domain is shown in SEQ ID NO: 11; More preferably, the anti-GPC3 / CD3 bispecific antigen binding molecule comprises a first peptide chain and a second peptide chain, the first peptide chain sequence is shown in SEQ ID NO: 12, and / or the second peptide chain sequence is shown in SEQ ID NO:

13. 2 . The pharmaceutical composition of claim 1 , wherein the concentration of the anti-GPC3 / CD3 bispecific antigen binding molecule is from about 1 mg / mL to about 100 mg / mL.

3. The pharmaceutical composition according to any one of claims 1 to 2, wherein the stabilizer is selected from one or more of sugars and amino acids.

4. The pharmaceutical composition according to claim 3, wherein the stabilizer is selected from one or more of sucrose, trehalose, sorbitol, mannitol, proline or a pharmaceutically acceptable salt, valine or a pharmaceutically acceptable salt, methionine or a pharmaceutically acceptable salt, phenylalanine or a pharmaceutically acceptable salt, leucine or a pharmaceutically acceptable salt, and glycine or a pharmaceutically acceptable salt. Optionally, the concentration of sucrose, trehalose, sorbitol, or mannitol is from about 20 mg / mL to about 100 mg / mL, and the concentration of proline or a pharmaceutically acceptable salt, valine or a pharmaceutically acceptable salt, methionine or a pharmaceutically acceptable salt, phenylalanine or a pharmaceutically acceptable salt, leucine or a pharmaceutically acceptable salt, and glycine or a pharmaceutically acceptable salt is from about 0 to about 100 mM.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the solubilizer is polysorbate 80 or polysorbate 20, preferably, the concentration of the solubilizer is about 0.1 mg / mL to about 3.0 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition further comprises a buffer selected from the group consisting of a histidine buffer, a succinate buffer, and a histidine-aspartate buffer, preferably, the concentration of the buffer is about 0 to about 20 mM.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pH of the pharmaceutical composition is about 4.0 to about 6.

0.

8. The pharmaceutical composition according to any one of claims 1 to 7, comprising the following components: (a) about 1 mg / mL to about 100 mg / mL of an anti-GPC3 / CD3 bispecific antigen binding molecule, (b) about 20 mg / mL to about 100 mg / mL of a saccharide and / or about 0 to about 100 mM of an amino acid, (c) about 0.1 mg / mL to about 3.0 mg / mL of polysorbate 80, and (d) about 0 to about 20 mM of a succinate buffer; the pH of the pharmaceutical composition is about 4.0 to about 6.

0.

9. A lyophilized preparation, which can form the pharmaceutical composition according to any one of claims 1 to 8 after being reconstituted. 10 . A lyophilized preparation obtained by freeze-drying the pharmaceutical composition according to claim 1 .

11. A method for preparing a lyophilized preparation, comprising the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 8.

12. Use of the pharmaceutical composition according to any one of claims 1 to 8 or the lyophilized preparation according to claim 9 or 10 in the preparation of a medicament for treating or preventing a disease associated with GPC3; preferably, the associated disease is cancer; more preferably, the cancer is selected from hepatocellular carcinoma, gastric cancer, esophageal cancer, ovarian cancer, or non-squamous non-small cell lung cancer.

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