Multispecific-antibody composition, preparation method therefor, and use thereof

Abstract

Provided is a composition which can be a lyophilized preparation solution or a lyophilized powder preparation. The composition comprises a multispecific antibody, a surfactant, a buffer system, and a stabilizer / osmotic pressure regulator. Further provided are a preparation method for the composition and use of the composition, for example, use in treating tumors, especially multiple myeloma.

Description

A multispecific antibody composition and its preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority and related rights of Chinese Patent Application No. 202311648214.5 filed on December 4, 2023, and the entire contents of the above-mentioned Chinese patent application are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of biomedicine, and in particular, to a multispecific antibody preparation, a preparation method, and an application thereof. Background Art

[0004] Multiple myeloma (MM) is a plasma cell malignancy characterized by the uncontrolled proliferation of plasma cells in the bone marrow, which acts like tumor cells and is accompanied by the secretion of monoclonal immunoglobulins. This can lead to a range of clinical manifestations, including multiple osteolytic lesions, hypercalcemia, anemia, renal damage, and recurrent infections. MM accounts for 1% of all cancers and 10-15% of hematologic malignancies. The male-to-female ratio is 1.6:1, and most patients are over 40 years old. Treatment for MM includes chemotherapy and hematopoietic stem cell transplantation.

[0005] B-cell maturation antigen (BCMA), also known as CD269 or TNFRSF17, is a member of the tumor necrosis factor receptor superfamily. It is primarily expressed on the surface of mature B lymphocytes and plasma cells and is a hallmark protein of B lymphocyte maturation. B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) are the primary ligands for BCMA, interacting with BCMA to transmit cell-stimulating signals, activating the TRAF-dependent NF-κB and JNK pathways, and increasing B-cell proliferation and survival. BCMA is primarily expressed on the surface of mature B lymphocytes and plasma cells and is rarely expressed in other tissue cells. BCMA is highly expressed in tumor cells in most patients with multiple myeloma and is an ideal therapeutic target for multiple myeloma.

[0006] GPRC5D is a G protein-coupled receptor C5 family subtype D, an orphan receptor and a seven-transmembrane protein. GPRC5D is a new target for multiple myeloma after BCMA. Tissue expression profiling studies have revealed that GPRC5D is specifically highly expressed in plasma cells of multiple myeloma, while its expression is low in normal tissues and restricted to immune-privileged hair follicles. Furthermore, GPRC5D expression does not overlap with BCMA. In tumor relapse models in which BCMA is lost, GPRC5D CAR-T remains effective, overcoming tumor escape. Theoretically, dual targeting of GPRC5D and BCMA could also cover a wider population and lead to better clinical responses.

[0007] The inventors of the present application have previously obtained a trispecific antibody molecule targeting BCMA-GPRC5D-CD3 and have demonstrated excellent anti-tumor activity. The relevant description is recorded in PCT application PCT / CN2023 / 098241 and is incorporated into the present application by reference in its entirety. Summary of the Invention

[0008] In a first aspect, the present disclosure provides a composition comprising a multispecific antibody molecule, wherein the multispecific antibody comprises:

[0009] (1) a first antigen-binding portion that binds to the BCMA antigen on tumor cells;

[0010] (2) a second antigen-binding portion that binds to the tumor cell GPRC5D antigen;

[0011] (3) A third antigen-binding portion that binds to the CD3 antigen on immune cells.

[0012] In some embodiments, the composition is a lyophilized preparation. In some embodiments, the composition comprises a multispecific antibody molecule, a buffer system, and a surfactant.

[0013] In certain embodiments, the composition comprises:

[0014] (1) Multispecific antibody molecules at approximately 1 mg / mL to 100 mg / mL;

[0015] (2) a buffer system of about 1 mM to 50 mM; and

[0016] (3) about 0.01% to 0.1% (w / v) of a surfactant;

[0017] Wherein, the pH value of the composition is not less than 5.0.

[0018] In certain embodiments, the composition has a pH of about 5.0 to 7.0.

[0019] In certain embodiments, the pH of the composition is about 5.0-6.0.

[0020] In certain embodiments, the pH of the composition is about 5.5.

[0021] In a preferred embodiment, the composition comprises:

[0022] (1) Multispecific antibodies;

[0023] (2) approximately 5-20 mM histidine-histidine hydrochloride buffer system;

[0024] (3) about 0.02% to 0.08% (w / v) polysorbate 80; and

[0025] (4) about 8.0% to 9.5% (w / v) trehalose;

[0026] Wherein, the pH of the composition is about 5.0 to 7.5.

[0027] In a more preferred embodiment, the composition comprises:

[0028] (1) Multispecific antibodies;

[0029] (2) approximately 5 mM histidine-histidine hydrochloride buffer system;

[0030] (3) about 0.04% (w / v) polysorbate 80; and

[0031] (4) about 9.5% (w / v) trehalose;

[0032] Wherein, the pH of the composition is about 5.5.

[0033] In certain embodiments, the first antigen-binding portion that binds to BCMA in the multispecific antibody comprises HCDR1, HCDR2, and HCDR3 of VHH, wherein the HCDR1 comprises the sequence shown in SEQ ID NO.2, the HCDR2 comprises the sequence shown in SEQ ID NO.3, and the HCDR3 comprises the sequence shown in SEQ ID NO.4.

[0034] In certain embodiments, the second antigen-binding portion that binds to GPRC5D in the multispecific antibody comprises a heavy chain variable region and a light chain variable region of an antibody or fragment thereof, wherein the heavy chain variable region comprises HCDR1 with a sequence as shown in SEQ ID NO.6, HCDR2 with a sequence as shown in SEQ ID NO.7, and HCDR3 with a sequence as shown in SEQ ID NO.8; and wherein the light chain variable region comprises LCDR1 with a sequence as shown in SEQ ID NO.10, LCDR2 with a sequence as shown in SEQ ID NO.11, and LCDR3 with a sequence as shown in SEQ ID NO.12.

[0035] In certain embodiments, the third antigen-binding portion of the multispecific antibody that binds to CD3 comprises a heavy chain variable region and a light chain variable region of an antibody or fragment thereof, wherein the heavy chain variable region comprises a HCDR1 with a sequence as shown in SEQ ID NO.14, a HCDR2 with a sequence as shown in SEQ ID NO.15, and a HCDR3 with a sequence as shown in SEQ ID NO.16; and wherein the light chain variable region comprises a LCDR1 with a sequence as shown in SEQ ID NO.18, a LCDR2 with a sequence as shown in SEQ ID NO.19, and a LCDR3 with a sequence as shown in SEQ ID NO.20.

[0036] In certain embodiments, the concentration of the multispecific antibody molecule is about 1 mg / mL to 100 mg / mL.

[0037] In certain embodiments, the concentration of the multispecific antibody molecule is about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL or about 100 mg / mL.

[0038] In certain embodiments, the concentration of the buffer system is about 1 mM to 50 mM.

[0039] In certain embodiments, the concentration of the buffer system is about 5 mM to 20 mM.

[0040] In certain embodiments, the concentration of the buffer system is about 5 mM, about 10 mM, about 15 mM, or about 20 mM.

[0041] In certain embodiments, the buffer system comprises one or more of acetate, citrate, histidine and / or phosphate buffer systems, preferably a histidine buffer system.

[0042] In certain embodiments, the buffer system comprises a histidine-histidine hydrochloride buffer system, and the concentration of the histidine-histidine hydrochloride buffer system is about 5 mM to 20 mM, preferably about 5 mM.

[0043] In certain embodiments, the content of the surfactant is 0.01% to 0.1% (w / v), preferably 0.02% to 0.08%, more preferably 0.02%, 0.04%, 0.06% or 0.08%.

[0044] In certain embodiments, the surfactant comprises polysorbate 80 and / or poloxamer 188.

[0045] In certain embodiments, the surfactant comprises polysorbate 80, and the content of polysorbate 80 is about 0.01% to 0.1%, preferably 0.04%.

[0046] In certain embodiments, the composition further comprises a protein stabilizer, wherein the protein stabilizer comprises sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol and / or sorbitol, preferably trehalose.

[0047] In certain embodiments, the concentration of the protein stabilizer is about 1% to 10% (w / v), preferably about 2% to 10%, more preferably about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9% or about 9.5%.

[0048] In certain embodiments, the protein stabilizer is trehalose, and the concentration of the trehalose is 2% to 10% (w / v), preferably 8% or 9.5%.

[0049] In certain embodiments, the composition further comprises an osmotic pressure regulating agent.

[0050] In certain embodiments, the osmotic pressure regulating agent comprises sodium chloride, potassium chloride, arginine hydrochloride, glycine, mannitol, sucrose, trehalose and / or sorbitol.

[0051] In certain embodiments, the composition further comprises a pH adjuster.

[0052] In certain embodiments, the pH adjuster includes acetic acid, sodium acetate, hydrochloric acid, sodium hydroxide, potassium hydroxide, citric acid, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and the like.

[0053] In a second aspect, the present disclosure provides a lyophilized powder formulation, which is prepared by freeze-drying the composition of the first aspect. In an optional embodiment, the composition is sterilized and / or pyrogen-free before freeze-drying.

[0054] In certain embodiments, the lyophilized powder formulation is reconstituted for subcutaneous injection in a patient.

[0055] In certain embodiments, the moisture content of the lyophilized powder formulation does not exceed 3%.

[0056] In certain embodiments, the concentration of the multispecific antibody after reconstitution of the lyophilized powder formulation is about 5 mg / mL to 100 mg / mL, preferably about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL or about 100 mg / mL.

[0057] In a third aspect, the present disclosure provides a method for preparing the composition of the first aspect (e.g., a lyophilized preparation), the method comprising the following steps:

[0058] (1) fully mixing the buffer component, the surfactant, and an auxiliary material such as a protein stabilizer with a solvent to obtain a mixed solution;

[0059] (2) thoroughly mixing the multispecific antibody molecule with the mixed solution;

[0060] Optionally, the preparation method further includes the steps of sterilization and heat source removal.

[0061] In the preparation method disclosed herein, there is no specific requirement for the order in which the buffer component, the surfactant, and the excipient are added, as long as the components are thoroughly mixed. The components can be mixed and fully dissolved in the solvent disclosed herein to form a uniform composition.

[0062] In a fourth aspect, the present disclosure further provides a method for preparing a lyophilized powder preparation, comprising freeze-drying the lyophilized preparation prepared according to the method described in the third aspect.

[0063] In a fifth aspect, the present disclosure further provides a lyophilized powder preparation for subcutaneous injection, wherein the lyophilized powder is prepared by freeze-drying the aforementioned lyophilized preparation solution.

[0064] In a sixth aspect, the present disclosure further provides use of the composition described in the first aspect, or the lyophilized powder preparation described in the second aspect or the fifth aspect, in the preparation of a drug for treating tumors.

[0065] In a seventh aspect, the present disclosure further provides use of the composition of the first aspect, or the lyophilized powder formulation of the second aspect or the fifth aspect, for treating a subject suffering from a tumor.

[0066] In an eighth aspect, the present disclosure further provides a method for treating a tumor in a subject, comprising administering the composition of the first aspect, or the lyophilized powder formulation of the second aspect or the fifth aspect to the subject.

[0067] In a ninth aspect, the present disclosure further provides the composition of the first aspect, or the lyophilized powder preparation of the second aspect or the fifth aspect, for use in treating tumors or treating subjects suffering from tumors.

[0068] In some embodiments, the tumor described in any one of the sixth to ninth aspects is a B-cell lymphoma. In a preferred embodiment, the B-cell lymphoma is multiple myeloma (MM). In a more preferred embodiment, the multiple myeloma is relapsed / refractory multiple myeloma (R / RMM).

[0069] In some embodiments, the present disclosure provides a lyophilized solution and lyophilized powder formulation for a large, asymmetric, multispecific antibody with excellent stability. In specific embodiments, the lyophilized powder formulation is stable for at least 24 months at 2-8°C, ensuring the efficacy of the large protein drug.

[0070] Because subcutaneous injection requires a small injection volume, the preparation of high-concentration, high-molecular-weight multispecific antibody formulations presents a challenge. The multispecific antibodies disclosed herein include a BCMA-GPRC5D-CD3 trispecific antibody with a large molecular weight. This disclosure provides a subcutaneous injection formulation comprising this trispecific antibody. This formulation is resistant to aggregation at high concentrations, both during preparation of the lyophilized formulation and upon reconstitution for administration, significantly improving patient compliance and / or drug accessibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 is a schematic diagram of the structure of the Tri-52 molecule.

[0072] Figure 2 is a trend chart of the SEC-HPLC results of the stability test of the final formulation.

[0073] Figure 3 is a trend chart of the CE-SDS (NR) results of the stability test of the final formulation.

[0074] FIG4 is a trend diagram of the CE-SDS(R) results of the stability test of the final formulation.

[0075] FIG5 is a trend chart of the iCIEF results of the stability test of the final formulation.

[0076] Detailed Description of the Invention

[0077] The preparations of macromolecular protein drugs (such as antibodies, etc.) are usually injections, which are suitable for non-intestinal administration, and their administration methods usually include subcutaneous, intravenous, intramuscular injections, etc. Since proteins are relatively unstable in solution and are very easy to form particles and aggregates, stability has become a difficult problem in the development of macromolecular protein drugs. The multispecific antibody (such as trispecific antibody) preparations disclosed in the present invention have a large molecular weight and complex structure of the main active ingredients, which puts higher requirements on the stability of the preparations. The development of preparations of macromolecular protein drugs (such as antibodies, etc.) first needs to solve the problem of protein stability. In order to solve the stability problem of multispecific antibodies (such as trispecific antibodies), the inventors of the present application have developed a lyophilized preparation liquid and a lyophilized powder preparation of multispecific antibodies, which achieves stable storage of multispecific antibodies (such as trispecific antibodies) at higher concentrations and can effectively prevent aggregation after redissolution. The preparations disclosed in the present invention can maintain the stability of multispecific antibodies during long-term storage and transportation of the product, thereby ensuring the stability, safety and / or effectiveness of the drug.

[0078] Definition of terms

[0079] Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings that are understood by those of ordinary skill in the art.

[0080] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0081] The terms "comprising," "including," and "having" are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also provides for "consisting of" solutions. For example, "a composition comprising A and B" should be understood to include the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0082] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the term."

[0083] The term "antibody" herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. "Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Differences in the amino acid composition and order of arrangement of the constant region of immunoglobulins' heavy chains result in varying antigenicity. Consequently, "immunoglobulins" can be classified into five classes, or isotypes, herein: IgM, IgD, IgG, IgA, and IgE, corresponding to their corresponding heavy chains: μ, δ, γ, α, and ε. Within the same class, Ig can be further divided into subclasses based on differences in the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

[0084] The "antibodies" herein also include antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0085] The term "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

[0086] The term "heavy chain antibody" herein refers to an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0087] The term "nanoantibody" in this article refers to heavy chain antibodies that are naturally missing light chains and exist in camels and other organisms. Cloning their variable regions can produce single-domain antibodies consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0088] The terms "VHH domain," "nanobody," and "single domain antibody" (sdAb) are synonymous and used interchangeably herein. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy chain variable region, obtained by cloning the variable region of a heavy chain antibody. This single-domain antibody (sdAb) is the smallest fully functional antigen-binding fragment. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting solely of a single heavy chain variable region.

[0089] The term "multi-specificity" herein refers to the ability of an antibody or antigen-binding fragment to specifically bind to, for example, different antigens or at least two different epitopes on the same antigen. Therefore, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes that an antibody can bind to. For example, conventional monospecific IgG antibodies have two identical antigen-binding sites (paratopes) and therefore can only bind to the same epitope (rather than binding to different epitopes). In contrast, multispecific antibodies have at least two different types of paratopes / binding sites and therefore can bind to at least two different epitopes. As described herein, "complementary determining region" refers to the antigen-binding site of an antibody. In addition, a single "specificity" can refer to one, two, three, or more than three identical complementary determining regions (the actual number of complementary determining regions / binding sites in a single antibody molecule is referred to as "valence") in a single antibody. For example, a single natural IgG antibody is monospecific and bivalent because it has two identical paratopes. Accordingly, a multispecific antibody comprises at least two (different) complementary determining regions / binding sites. Therefore, the term "multispecific antibody" refers to an antibody having more than one paratope and having the ability to bind to two or more different epitopes. The term "multispecific antibody" specifically includes bispecific antibodies as defined above, but generally also includes proteins, such as antibodies that specifically bind to three or more different epitopes, scaffolds, i.e., antibodies with three or more paratopes / binding sites. Exemplarily, the multispecific antibody included in the composition of the present disclosure is a trispecific antibody targeting BCMA-GPRC5D-CD3, the structure of which is shown in Figure 1.

[0090] The term "valent" herein refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites, and six binding sites, respectively, in an antibody / antigen-binding molecule. Exemplarily, the trispecific antibody comprised in the compositions of the present disclosure is tetravalent and comprises one binding site that binds BCMA, two binding sites that bind GPRCC5D, and one binding site that binds CD3.

[0091] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0092] The term "variable region" herein refers to the region of an antibody heavy or light chain involved in antigen binding. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable regions of natural antibody heavy and light chains generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).

[0093] The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) found in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT), while being considered to be outside the hypervariable region under a different set of criteria (such as KABAT or IMGT). One or more of these positions can also be found in an extended hypervariable region. The present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region can be abbreviated as LCDRs. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3), which form a loop connecting the sheet structure and, in some cases, form a portion of the sheet structure. The CDRs in each chain are held together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and contribute to the formation of the antibody antigen-binding site with the CDRs from other antibody chains.

[0094] "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0095] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, and has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" can be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. A "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to that of a native antibody constant region, while the latter only includes "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from an Fc or CH3 domain.

[0096] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0097] The term "Fc region" herein is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. For example, the human IgG heavy chain Fc region may extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage, removing one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells through expression of a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may be present or absent. Typically, an IgG Fc region comprises an IgG CH2 and IgG CH3 domains, and optionally, may further comprise a complete or partial hinge region, but does not comprise a CH1 domain. The "CH2 domain" of a human IgG Fc region typically extends from an amino acid residue at approximately position 231 to an amino acid residue at approximately position 340. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index.

[0098] The term "Fc variant" herein refers to changes in Fc structure or function caused by one or more amino acid substitutions, insertions, or deletions at appropriate sites on the Fc protein. "Inter-Fc variant interactions" refer to interactions between Fc variants engineered through mutations that can form space-filling effects, electrostatic guidance, hydrogen bonding, hydrophobic interactions, and the like. Interactions between Fc variants contribute to the formation of stable heterodimeric proteins. A preferred mutation design is a "knob-into-hole (KIH)" mutation design.

[0099] Mutational design techniques for Fc variants have been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include the "knob-into-hole" (KIH) approach proposed by Cater et al.; the electrostatic steering approach to form Fc-containing heterodimers by Amgen (US 20100286374A1); the SEEDbodies approach formed by IgG / IgA chain exchange proposed by Jonathan H. Davis et al.; bispecific molecules formed using Genmab's DuoBody platform technology; Xencor's approach to heterodimeric protein formation using a combination of structural calculations and Fc amino acid mutations, integrating different modes of action; and the charge network-based Fc engineering approach from Suzhou Alphamab (CN201110459100.7) to generate heterodimeric proteins. Other genetic engineering approaches, such as those based on Fc amino acid changes or functional modifications, have been proposed to generate functional heterodimeric proteins. The Knob / Hole structure on the Fc variant fragment disclosed in the present invention refers to the mutation of each of the two Fc fragments, which can be combined in the form of "Knob-into-Hole" after the mutation. Preferably, the "knob-into-hole" model of Cater et al. is used to perform site mutation transformation on the Fc region, so that the first Fc variant and the second Fc variant obtained can be combined together in the form of "knob-into-hole" to form a heterodimer. It is within the scope of those skilled in the art to select a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass. Preferably, the Fc region of human antibodies IgG1, IgG2, IgG3, and IgG4, more preferably the Fc region of human antibody IgG1. Randomly select one of the first Fc variant or the second Fc variant to make a knob mutation and the other to make a hole mutation.

[0100] The term "effector function" herein refers to the activity of an antibody molecule, which is mediated by the binding of the domain of the antibody rather than the antigen binding domain, typically mediated by the binding of effector molecules. Effector function includes complement-mediated effector function, which is mediated by the binding of, for example, the C1 component of the complement to the antibody. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and can participate in autoimmune hypersensitivity responses. Effector function also includes Fc receptor (FcR)-mediated effector function, which can be triggered by the binding of the constant domain of the antibody to the Fc receptor (FcR). The binding of antibodies to Fc receptors on the cell surface triggers many important and various biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, dissolution of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. The effector function of an antibody can be altered by changing, for example, enhancing or reducing the affinity of the antibody for effector molecules such as Fc receptors or complement components. Binding affinity will typically be altered by modifying the effector molecule binding site, and in this case, it is appropriate to locate the site of interest and modify at least a portion of the site in a suitable manner. It is also envisioned that changes in the binding site on an antibody for an effector molecule need not significantly alter the overall binding affinity, but may alter the geometry of the interaction, rendering the effector mechanism ineffective, as in non-productive binding. It is further envisioned that the effector function can also be altered by modifying sites that are not directly involved in effector molecule binding but that otherwise participate in the performance of the effector function.

[0101] The term "identity" herein can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. The percentage of identity between the two sequences varies with the number of identical positions shared by the sequences, taking into account the number of spaces that need to be introduced and the length of each space for optimal comparison of the two sequences.

[0102] Mathematical algorithms can be used to compare sequences between two sequences and calculate percent identity. For example, the Needlema and Wunsch algorithms (available at www.gcg.com) that have been integrated into the GAP program of the GCG software package are used, using a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program (available at www.gcg.com) in the GCG software package is used, using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0103] The terms "linker," "connector," and "linker" are used interchangeably herein to refer to a connecting polypeptide sequence used to connect protein domains. The linker typically possesses a degree of flexibility, and its use does not compromise the original functionality of the protein domains. Exemplary linkers include (G4S)3.

[0104] The terms "composition" and "formulation" herein refer to a product comprising the multispecific antibodies described herein in predetermined amounts or ratios, as well as any product produced directly or indirectly by combining predetermined amounts of the multispecific antibodies described herein. The compositions or formulations described herein may include pharmaceutical formulations, i.e., comprising the multispecific antibodies described herein and excipients, buffer components, surfactants, and any product produced directly or indirectly by combining, complexing, or aggregating any two or more of these components. The formulations may be in the form of solutions or lyophilized powders.

[0105] The term "buffer component" herein refers to an agent that provides a buffering effect (e.g., to resist changes in pH). For example, the buffer component can regulate changes in pH caused by the addition and / or release of acidic or basic substances. For example, the buffer component can include a weak acid and its conjugate base; or a weak base and its conjugate acid.

[0106] As used herein, "buffer" or "buffer solution" generally refers to an aqueous solution comprising a mixture of an acid (usually a weak acid, such as acetic acid, citric acid, or histidine hydrochloride) and its conjugate base (e.g., acetate or citrate, such as sodium acetate, sodium citrate, or histidine), or a mixture of a base (usually a weak base, such as histidine) and its conjugate acid (e.g., a protonated histidine salt). Due to the "buffering effect" imparted by the "buffering agent," the pH of the "buffer solution" changes only slightly when a small amount of strong acid or base is added.

[0107] The term "pH adjuster" herein includes substances that adjust pH. The term "pH adjuster" also refers to multiple pH adjusters. The term "pH adjuster" refers to one pH adjuster or two or more pH adjusters. Therefore, the term "pH adjuster" also includes mixtures containing or consisting of different pH adjusters. Examples of pH adjusters include acetic acid, sodium acetate, hydrochloric acid, sodium hydroxide, potassium hydroxide, citric acid, sodium citrate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0108] The term "surfactant" herein refers to an agent that can protect a protein (e.g., a multispecific antibody of the present disclosure) from air / solution interface-induced stress, solution / surface-induced stress, to reduce aggregation of the protein or minimize the formation of particulate matter in the composition. The surfactant can contain both a hydrophobic group and a hydrophilic group. The surfactant can include a mixture or combination of one or more surfactants. The surfactant can include a nonionic or ionic surfactant.

[0109] The term "nonionic surfactant" herein refers to a surfactant containing a hydrophilic group that does not dissociate in an aqueous solution. For example, the nonionic surfactant may not dissociate into an ionic state in an aqueous solution, but may exist in the solution in a molecular or micellar state. For example, the nonionic surfactant may include a polyoxyethylene nonionic surfactant. (For example, fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether (OP), octylphenol polyoxyethylene ether-10, dodecylphenol polyoxyethylene ether or fatty acid polyoxyethylene ether), a polyol-type inactive surfactant (for example, a span type surfactant, a tween type surfactant, polysorbate) and / or a polyether (for example, a fully embedded polyether). The nonionic surfactant may have high qualitative properties, be less affected by acids, alkalis, and ions, and / or have strong hard water resistance.

[0110] The term "osmotic pressure regulator" herein refers to an agent for regulating the osmotic pressure of a liquid preparation. For example, the osmotic pressure regulator can make the osmotic pressure of the liquid preparation comprising it isotonic with body fluids (e.g., human plasma) to avoid damaging tissue. Examples of osmotic pressure regulators herein include sodium chloride, potassium chloride, arginine hydrochloride, glycine, mannitol, sucrose, trehalose, and / or sorbitol.

[0111] In the present disclosure, the term "solvent" generally refers to a liquid having the ability to dissolve other substances. The solvent may include organic solvents (such as aromatic hydrocarbons, aliphatic hydrocarbons, alicyclic hydrocarbons, halogenated hydrocarbons, alcohols, ethers, esters or ketones) and inorganic solvents (such as water). For example, the water may include pure water, i.e., H2O without impurities. For example, the water may include water for injection, i.e., water that meets the requirements of the Chinese Pharmacopoeia Water for Injection. The water for injection may include distilled water or deionized water obtained by distillation.

[0112] In the present disclosure, the terms "stability" and "stable" refer to the resistance of the antibody in the formulation to degradation or fragmentation under given manufacturing, preparation, transportation and storage conditions. A "stable" formulation retains biological activity under given manufacturing, preparation, transportation and storage conditions. Compared with a reference formulation, such as by appearance, pH, concentration, purity (molecular exclusion chromatography (SEC-HPLC), non-reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS (NR), reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS (R)), charge isomerism (full column capillary isoelectric focusing (iCIEF)), dynamic light scattering (DLS) detection of particle size, binding activity (ELISA test). The detection method is generally carried out in accordance with the latest edition of the Chinese Pharmacopoeia and corresponding guidelines.

[0113] In the present disclosure, the term "about" refers to a variation within the range of 0.5%-10% above or below the specified value, for example, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% above or below the specified value. Exemplarily, for surfactants, the concentration is about 0.01% to 0.1%, and the percentage values ​​are allowed to be within the error range allowed in the art, such as ±0.01%; for protein stabilizers, the concentration is about 1% to 10%, and the percentage values ​​are allowed to be within the error range allowed in the art, such as ±0.5%.

[0114] In the present disclosure, when referring to the concentration or content of surfactants, excipients such as stabilizers, etc., it refers to the mass concentration, which can also be expressed as w / v.

[0115] The term "administer" and similar terms herein are generally not limited to physical administration, and suitable methods include in vitro, ex vivo, or in vivo methods. For example, any method of administration known to those skilled in the art for contacting cells, organs, or tissues with a composition can be used. For example, the compound can be introduced into the body of a subject in need of treatment by any introduction or delivery route. In some embodiments, the compositions of the present disclosure are administered primarily subcutaneously.

[0116] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0117] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Exemplarily, a "subject" includes a mammal, such as a human, primate (e.g., monkey), or non-primate mammal, being treated for a disease or condition.

[0118] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0119] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the term "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0120] The present disclosure will be further described below with reference to specific implementation plans and examples, and the advantages and features of the present disclosure will become clearer as the description proceeds.

[0121] Composition

[0122] In one aspect, the present disclosure provides a composition that is a lyophilized preparation comprising a multispecific antibody, a buffer component, and a surfactant.

[0123] In some embodiments, the composition comprises the multispecific antibody at a concentration of about 1 mg / mL to 100 mg / mL.

[0124] In some embodiments, the concentration of the multispecific antibody is about 5 mg / mL to 100 mg / mL.

[0125] In some embodiments, the concentration of the multispecific antibody is about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL or about 100 mg / mL, or any concentration in between any two of the foregoing values.

[0126] In some embodiments, the multispecific antibody comprises:

[0127] (1) a first antigen-binding portion that binds to the BCMA antigen on tumor cells;

[0128] (2) a second antigen-binding portion that binds to the tumor cell GPRC5D antigen;

[0129] (3) A third antigen-binding portion that binds to the CD3 antigen on immune cells.

[0130] In some embodiments, the multispecific antibody comprises a first light chain, a second light chain, a first heavy chain, and a second heavy chain, wherein:

[0131] (1) The first light chain and the second light chain are identical and have the following structure: VL a -CL; and

[0132] (2) The first heavy chain and the second heavy chain are different and have the following structure: The first heavy chain comprises VHH b -L1-VH a -CH1-Fc1 and a second heavy chain containing VH a -CH1-L2-VH CD3 -L3-VL CD3 -Fc2; optionally, one heavy chain further carries a Flag tag at the carboxyl terminus and / or the other heavy chain further carries a His tag at the carboxyl terminus;

[0133] Among them VH a and VL a The heavy chain variable region and light chain variable region that specifically bind to GPRC5D, VHH b is a nanobody that specifically binds to BCMA, VH CD3 and VL CD3 They are the heavy chain variable region and light chain variable region that specifically bind to CD3, and L1, L2, and L3 are the same or different linkers.

[0134] In some embodiments, the structure of the first heavy chain is VHH b -L1-VH a -CH1-Fc1. ​​In some embodiments, the structure of the second heavy chain is VH a -CH1-L2-VH CD3 -L3-VL CD3 -Fc2.

[0135] In some embodiments, the Fc1 and Fc2 of the multispecific antibody are different, Fc1 is knob-Fc, and Fc2 is hole-Fc; or, Fc1 is hole-Fc, and Fc2 is knob-Fc; preferably, wherein the knob-Fc comprises a T366W mutation, and / or the hole-Fc comprises T366S, L368A and / or Y407V mutations; preferably, Fc1 and Fc2 are the Fc regions of IgG4; more preferably, Fc1 is knob-Fc, and Fc2 is hole-Fc.

[0136] In some embodiments, the Fc1 and Fc2 of the multispecific antibody are different and have amino acid mutations that alter effector function. In a preferred embodiment, the mutations that alter effector function comprise S228P, F234A, and / or L235A mutations of IgG4 Fc.

[0137] In some embodiments, for the multispecific antibody:

[0138] (1) The first antigen-binding portion targeting BCMA comprises HCDR1, HCDR2, and HCDR3 of VHH, wherein the HCDR1 comprises the sequence shown in SEQ ID NO.2, the HCDR2 comprises the sequence shown in SEQ ID NO.3, and the HCDR3 comprises the sequence shown in SEQ ID NO.4;

[0139] (2) the second antigen-binding portion targeting GPRC5D comprises a heavy chain variable region and a light chain variable region of an antibody or fragment thereof, wherein the heavy chain variable region comprises HCDR1 of SEQ ID NO.6, HCDR2 of SEQ ID NO.7, and HCDR3 of SEQ ID NO.8; and the light chain variable region comprises LCDR1 of SEQ ID NO.10, LCDR2 of SEQ ID NO.11, and LCDR3 of SEQ ID NO.12; and

[0140] (3) The third antigen-binding portion targeting CD3 comprises a heavy chain variable region and a light chain variable region of an antibody or a fragment thereof, wherein the heavy chain variable region comprises HCDR1 with a sequence as shown in SEQ ID NO.14, HCDR2 with a sequence as shown in SEQ ID NO.15, and HCDR3 with a sequence as shown in SEQ ID NO.16; and the light chain variable region comprises LCDR1 with a sequence as shown in SEQ ID NO.18, LCDR2 with a sequence as shown in SEQ ID NO.19, and LCDR3 with a sequence as shown in SEQ ID NO.20.

[0141] In some embodiments, for the multispecific antibody:

[0142] (1) The first antigen-binding portion targeting BCMA comprises a VHH sequence as shown in SEQ ID NO.1;

[0143] (2) the second antigen-binding portion targeting GPRC5D comprises a heavy chain variable region having a sequence as shown in SEQ ID NO. 5 and a light chain variable region having a sequence as shown in SEQ ID NO. 9; and

[0144] (3) The third antigen-binding portion targeting CD3 comprises a heavy chain variable region with a sequence as shown in SEQ ID NO.13 and a light chain variable region with a sequence as shown in SEQ ID NO.17.

[0145] In some embodiments, the multispecific antibody comprises the Fc domain of human IgG4.

[0146] In some embodiments, the human IgG4 Fc domain comprises distinct Fc1 and Fc2.

[0147] In some embodiments, the different Fc1 and Fc2 comprise amino acid mutations that alter effector function, wherein the mutations that alter effector function comprise S228P, F234A, and / or L235A mutations of IgG4 Fc.

[0148] In some embodiments, the different Fc1 and Fc2 regions comprise heterodimerization-promoting amino acid mutations, wherein the heterodimerization-promoting amino acid mutations comprise knob-into-hole (KIH) mutations. In preferred embodiments, the knob-into-hole mutations comprise: (a) either Fc region comprises a T366W mutation; and (b) the other Fc region comprises a T366S, L368A, and / or Y407V mutation.

[0149] In some embodiments, the multispecific antibody is a trispecific antibody. In some embodiments, the trispecific antibody comprises a first heavy chain having a sequence as shown in SEQ ID NO. 21, a second heavy chain having a sequence as shown in SEQ ID NO. 22, a first light chain having a sequence as shown in SEQ ID NO. 23, and a second light chain having a sequence as shown in SEQ ID NO. 23.

[0150] In some embodiments, either Fc region further comprises a His-tag, and the other Fc region further comprises a Flag-tag.

[0151] In some embodiments, the pH of the composition is about 5.0-7.0.

[0152] In some embodiments, the pH of the composition is about 5.0 to 6.5.

[0153] In some embodiments, the pH of the composition is about 5.0-6.0.

[0154] In some embodiments, the pH of the composition is about 5.5-6.0.

[0155] In some embodiments, the pH of the composition is about 5.5.

[0156] In some embodiments, the buffer component comprises a citrate buffer solution, a histidine buffer solution and / or a phosphate buffer solution. In a preferred embodiment, the buffer component is a histidine buffer solution, such as a histidine-histidine hydrochloride buffer solution.

[0157] In some embodiments, the buffer component content is about 1-50 mM, about 1-45 mM, about 1-40 mM, about 1-35 mM, about 1-20 mM, about 1-15 mM, about 1-10 mM, about 1-5 mM, about 5-50 mM, about 5-45 mM, about 5-40 mM, about 5-35 mM, about 5-30 mM, about 5-25 mM or about 5-20 mM.

[0158] In some embodiments, the buffer component comprises a histidine-histidine hydrochloride buffer solution, and the content of the histidine-hydrochloride buffer solution can be about 1-50 mM, about 1-45 mM, about 1-40 mM, about 1-35 mM, about 1-20 mM, about 1-15 mM, about 1-10 mM, about 1-5 mM, about 5-50 mM, about 5-45 mM, about 5-40 mM, about 5-35 mM, about 5-30 mM, about 5-25 mM, or about 5-20 mM. In a preferred embodiment, the content of the histidine-hydrochloride buffer solution is about 5-20 mM, more preferably 5 mM.

[0159] In some embodiments, the buffer component content is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or any content between any two of the above values.

[0160] In some embodiments, the buffer component includes a histidine-histidine hydrochloride buffer solution, and the content of the histidine-hydrochloric acid buffer solution can be about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or any content between any two of the above values.

[0161] In some embodiments, the surfactant comprises polysorbate 80 and / or poloxamer 188. In a preferred embodiment, the surfactant is polysorbate 80.

[0162] In some embodiments, the surfactant comprises polysorbate 80, and the mass concentration of the polysorbate 80 can be about 0.01% to 1%, about 0.01 to 0.5%, about 0.01 to 0.1%, about 0.01 to 0.08%, about 0.01 to 0.06%, about 0.01 to 0.04%, about 0.01 to 0.02%, about 0.02 to 1%, about 0.04 to 1%, about 0.02 to 0.5%, about 0.02 to 0.1% or about 0.02 to 0.08%. In a preferred embodiment, the mass concentration of the polysorbate 80 is about 0.02 to 0.08%, more preferably 0.04%.

[0163] In some embodiments, the surfactant comprises polysorbate 80, and the mass concentration of polysorbate 80 can be about 0.2%, 0.4%, 0.6% or 0.8%, or any content between any two of the above values.

[0164] In some embodiments, the surfactant comprises poloxamer 188, and the mass concentration of poloxamer 188 can be about 0.01% to 1%, about 0.01 to 0.5%, about 0.01 to 0.1%, about 0.01 to 0.08%, about 0.01 to 0.06%, about 0.01 to 0.04%, about 0.01 to 0.02%, about 0.02 to 1%, about 0.04 to 1%, about 0.02 to 0.5%, about 0.02 to 0.1% or about 0.02 to 0.08%.

[0165] In some embodiments, the surfactant comprises poloxamer 188, and the mass concentration of poloxamer 188 can be about 0.2%, 0.4%, 0.6% or 0.8%, or any content between any two of the above values.

[0166] In some embodiments, the composition further comprises an excipient. In a preferred embodiment, the excipient is a stabilizer, such as a protein stabilizer.

[0167] In some embodiments, the composition further comprises a stabilizer. For example, the stabilizer comprises glycine, sucrose, trehalose and / or mannitol. In a preferred embodiment, the stabilizer is trehalose or sucrose.

[0168] In a more preferred embodiment, the stabilizer is trehalose. In some embodiments, trehalose can improve the stability of the formulation within a reasonable concentration range, so trehalose is the most preferred excipient.

[0169] In some embodiments, the mass concentration of the stabilizer is about 1% to 10%, about 1% to 9.5%, about 1% to 9.0%, about 1% to 8.5%, about 1% to 8.0%, about 1% to 7.5%, about 1% to 7.0%, about 1% to 6.5%, about 1% to 6.0%, about 1% to 5.5%, about 1% to 5.0%, about 1% to 4.5%, about 1% to 4%, about 1% to 3.5%, about 1% to 3%, about 1% to 2.5%, about 1% to 2%, about 2% to 10%, about 2% to 9.5%, about 2% to 9.0%, about 2% to 8.5%, about 2% to 8.0%, about 2% to 7.5%, about 2% to 7.0%, about 2% to 6.5%, about 2% to 6.0%, about 2% to 5.5%, about 2% to 5.0% or about 2% to 4.5%.

[0170] In some embodiments, the mass concentration of the stabilizer is about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5% or about 10%, or any content between any two of the above values.

[0171] In some embodiments, the stabilizer comprises trehalose, and the mass concentration of the trehalose is about 1% to 10%, about 1% to 9.5%, about 1% to 9.0%, about 1% to 8.5%, about 1% to 8.0%, about 1% to 7.5%, about 1% to 7.0%, about 2% to 10%, about 2% to 9.5%, about 2% to 9.0%, about 2% to 8.5%, about 2% to 8.0%, about 2% to 7.5%, about 2% to 7.0%, about 4.5% to 10%, about 4.5% to 9.5%, about 4.5% to 9.0%, about 4.5% to 8.5%, about 4.5% to 8.0%, about 4.5% to 7.5% or about 4.5% to 7.0%. In a preferred embodiment, the mass concentration of the trehalose is about 8.0% to 9.5%, more preferably 9.5%.

[0172] In some embodiments, the stabilizer comprises trehalose, and the mass concentration of the trehalose is about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5% or about 10%, or any content between any two of the above values.

[0173] In some embodiments, the stabilizer comprises glycine, and the mass concentration of the glycine is about 1% to 5.0%, about 1% to 4.5%, about 1% to 4.0%, about 1% to 3.5%, about 1% to 3.0%, about 1% to 2.5%, about 1% to 2.0%, about 1.5% to 5.0%, about 1.5% to 4.5%, about 1.5% to 4.0%, about 1.5% to 3.5%, about 1.5% to 3.0%, about 1.5% to 2.5% or about 1.5% to 2.0%.

[0174] In some embodiments, the stabilizer comprises glycine, and the glycine content can be about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5% or about 10%.

[0175] In some embodiments, the stabilizer comprises sucrose, and the mass concentration of the sucrose is about 1% to 10%, about 1% to 9.5%, about 1% to 9.0%, about 1% to 8.5%, about 1% to 8.0%, about 1% to 7.5%, about 1% to 7.0%, about 2% to 10%, about 2% to 9.5%, about 2% to 9.0%, about 2% to 8.5%, about 2% to 8.0%, about 2% to 7.5%, about 2% to 7.0%, about 4.5% to 10%, about 4.5% to 9.5%, about 4.5% to 9.0%, about 4.5% to 8.5%, about 4.5% to 8.0%, about 4.5% to 7.5% or about 4.5% to 7.0%.

[0176] In some embodiments, the stabilizer comprises sucrose, and the sucrose content is about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5% or about 10%, or any amount in between any two of the above values.

[0177] In some embodiments, the stabilizer comprises mannitol, and the mass concentration of the mannitol is about 1% to 10%, about 1% to 9.5%, about 1% to 9.0%, about 1% to 8.5%, about 1% to 8.0%, about 1% to 7.5%, about 1% to 7.0%, about 1% to 6.5%, about 1% to 6.0%, about 1% to 5.5%, about 1% to 5.0%, about 1% to 4.5%, about 2% to 10%, about 2% to 9.5%, about 2% to 9.0%, about 2% to 8.5%, about 2% to 8.0%, about 2% to 7.5%, about 2% to 7.0%, about 2% to 6.5%, about 2% to 6.0%, about 2% to 5.5%, about 2% to 5.0% or about 2% to 4.5%.

[0178] In some embodiments, the stabilizer comprises mannitol, and the mass concentration of the mannitol can be about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5% or about 10%, or any content between any two of the above values.

[0179] In some embodiments, the composition comprises:

[0180] (1) Multispecific antibodies;

[0181] (2) approximately 5-20 mM histidine-histidine hydrochloride buffer system;

[0182] (3) polysorbate 80 at a concentration of approximately 0.02% to 0.08% by mass; and

[0183] (4) Trehalose at a concentration of approximately 8.0% to 9.5% by mass;

[0184] Wherein, the pH of the composition is about 5.0 to 7.5.

[0185] In some embodiments, the composition comprises:

[0186] (1) Multispecific antibodies;

[0187] (2) approximately 5 mM histidine-histidine hydrochloride buffer system;

[0188] (3) approximately 0.04% by mass of polysorbate 80; and

[0189] (4) Trehalose at a concentration of approximately 9.5% by mass;

[0190] Wherein, the pH of the composition is about 5.5.

[0191] In some embodiments, the composition comprises:

[0192] (1) Multispecific antibodies at approximately 1 mg / mL to 100 mg / mL;

[0193] (2) about 5 to 20 mM buffer component;

[0194] (3) a surfactant at a concentration of about 0.02% to 0.08% by mass; and

[0195] (4) Stabilizer at a concentration of approximately 1% to 10% by mass;

[0196] The pH of the composition may be about 5.0 to 7.5.

[0197] In some embodiments, the composition comprises:

[0198] (1) Multispecific antibodies at approximately 1 mg / mL to 100 mg / mL;

[0199] (2) approximately 5-20 mM histidine-histidine hydrochloride buffer system;

[0200] (3) polysorbate 80 at a concentration of approximately 0.02% to 0.08% by mass; and

[0201] (4) Trehalose at a concentration of approximately 1% to 10% by mass;

[0202] The pH of the composition may be about 5.0 to 7.5.

[0203] In some embodiments, the composition comprises:

[0204] (1) approximately 10 mg / mL of multispecific antibodies;

[0205] (2) approximately 5 mM histidine-histidine hydrochloride buffer system;

[0206] (3) about 0.04% (w / v) polysorbate 80; and

[0207] (4) about 9.5% (w / v) trehalose;

[0208] Wherein, the pH of the composition is about 5.5.

[0209] In some embodiments, the composition further comprises a solvent. For example, the solvent comprises sterile purified water and / or water for injection.

[0210] On the other hand, the present disclosure provides a lyophilized powder preparation, wherein the lyophilized powder is obtained by freeze-drying the aforementioned lyophilized preparation liquid; optionally, the freeze-drying step further includes sterilizing and removing pyrogens from the lyophilized preparation liquid.

[0211] In some embodiments, the lyophilized powder formulations described herein are reconstituted for subcutaneous administration to a patient.

[0212] Preparation method

[0213] In one aspect, the present disclosure provides a method for preparing the aforementioned lyophilized preparation, the method comprising the following steps:

[0214] (1) fully mixing the buffer component, the surfactant, and the excipient with a solvent to obtain a mixed solution; and

[0215] (2) thoroughly mixing the multispecific antibody molecule with the mixed solution;

[0216] Optionally, the preparation method further includes the steps of sterilization and heat source removal.

[0217] In the preparation method disclosed herein, there is no specific requirement for the order in which the buffer component, the surfactant, and the excipient are added, as long as the components are thoroughly mixed. The components can be mixed and fully dissolved in the solvent disclosed herein to form a uniform composition.

[0218] On the other hand, the present disclosure also provides a method for preparing a lyophilized powder preparation, comprising freeze-drying the lyophilized preparation obtained above.

[0219] Therapeutic uses

[0220] The present disclosure provides a use of the composition or lyophilized powder preparation described in the present disclosure in preparing a drug for treating tumors.

[0221] The present disclosure also provides use of the composition or lyophilized powder formulation of the present disclosure for treating a subject suffering from a tumor.

[0222] In some embodiments, the tumor is a B cell lymphoma. In a preferred embodiment, the B cell lymphoma is multiple myeloma (MM). In a more preferred embodiment, the multiple myeloma is relapsed / refractory multiple myeloma (R / RMM). Example

[0223] The embodiments disclosed herein are merely exemplary and do not constitute any limitation to the scope of the present disclosure. It should be understood by those skilled in the art that the details and form of the disclosed technical solution can be modified or replaced without departing from the spirit and scope of the present disclosure, but these modifications and replacements all fall within the scope of protection of the present disclosure. In the embodiments, if specific conditions are not specified, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0224] In the present disclosure, the BCMA-GPRC5D-CD3 multispecific antibody is as disclosed in PCT / CN2023 / 098241, which is incorporated herein by reference in its entirety.

[0225] Example 1. Preparation of trispecific antibodies

[0226] 1.1 Discovery of tri-antibody molecules

[0227] In previous studies, a trispecific antibody Tri-52 (PCT / CN2023 / 098241) targeting BCMA, GPRC5D, and CD3 has been developed. The structure is shown in Figure 1 , and the CDR and variable region sequences of the BCMA, GPRC5D, and CD3 targeting portions are shown in Table 1 . The CDRs are defined according to Kabat numbering.

[0228] Table 1 Antibody molecule amino acid sequence

[0229] 1.2 Expression and purification of trispecific antibodies

[0230] The plasmid encoding the antibody gene was transfected into Expi293F cells (Thermofisher, Catalog No. A14527). Six days after transfection, the cell supernatant was collected and purified using AKTA Pure Protein A affinity purification and molecular sieve purification. The protein was then sterile-filtered using a 0.22μm filter. The resulting antibody was quantitatively and qualitatively analyzed by SDS-PAGE, SEC-HPLC, and CE-SDS, yielding a highly pure Tri-52 trispecific antibody molecule.

[0231] 1.3 Trispecific Antibody Binding Experiment

[0232] FACS was used to identify the binding activity of the three-antibody molecule to tumor cells endogenously expressing BCMA and GPRC5D. The positive control antibodies were Regeneron's bispecific antibody REGN5459 (WO2020018820A1, targeting BCMA antigen and CD3 receptor expressed on the surface of T cells) and Johnson & Johnson's bispecific antibody JNJ7564 (WO2019220368A1, targeting GPRC5D antigen and CD3 receptor expressed on the surface of T cells). The negative control antibody was anti-FITC×CD3-Hab01 (prepared in-house). The results are shown in Table 2. The results showed that the three-antibody molecule can strongly bind to the endogenous tumor cell NCI-H929 expressing the antigens BCMA and GPRC5D.

[0233] Table 2 FACS detection of the binding reaction between the three antibodies and H929 cells

[0234] Example 2. Buffer system screening

[0235] 2.1 Buffer system screening plan

[0236] The screening experiment covered three buffer salts (citrate, histidine, and phosphate) and nine formulation buffer systems (pH ranging from 5.0 to 7.5). Tri-52 protein was used in each formulation at a protein concentration of 10 mg / mL. The protein stock solution was ultrafiltration, concentrated, and replaced in the corresponding nine buffer systems. The protein was tested using a 40°C high-temperature accelerated test. The stability of the protein in the nine buffer systems was comprehensively evaluated and compared using appearance, pH, concentration, purity (size exclusion chromatography (SEC-HPLC), non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS(NR), reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS(R))), charge isomerism (full-column capillary isoelectric focusing (iCIEF)), dynamic light scattering (DLS) to detect particle size, and activity (ELISA to detect binding activity) as indicators to screen for the optimal buffer system. The specific formulation composition and investigation plan are shown in Table 3.

[0237] The relevant detection methods for various indicators of the preparations disclosed in the present invention are all conventional detection methods well known to those skilled in the art, for example, referring to the standards of Part III of the "Chinese Pharmacopoeia (2020 Edition)" and relevant guidelines.

[0238] Table 3 Experimental plan for screening formulation buffer systems Note: X = appearance, pH, concentration, iCIEF, SEC-HPLC, CE-SDS (NR), CE-SDS (R), DLS, activity. T0 = starting point of investigation; 1W = 1 week, and so on.

[0239] 2.2 Buffer system screening results

[0240] The main results of buffer system screening are summarized in Tables 4, 5 and 6.

[0241] Table 4 Preparation buffer system screening 40℃ accelerated test results 1 Note: T0 = starting point of investigation; N / A = not applicable

[0242] Table 5 Preparation buffer system screening 40℃ accelerated test results 2 Note: T0 = investigation starting point; N / A = not applicable; ND = not detected

[0243] Table 6 Preparation buffer system screening 40 ° C accelerated test results 3 Note: T0 = starting point of investigation; N / A = not applicable

[0244] 2.3 Buffer system screening conclusion

[0245] This buffer system screening experiment covered three buffer salts (citrate, histidine, and phosphate) and nine formulation buffer systems (pH values ​​ranging from 5.0 to 7.5). A 40°C high-temperature accelerated test was conducted to comprehensively compare the stability of this protein in the nine buffer systems. The results are as follows:

[0246] Appearance: When tested at 40°C from T0 to 4 weeks, F1-F3 (citrate buffer system) and F7-F9 (phosphate buffer system) showed a large amount of precipitation. F4-F6 (histidine buffer system) changed from a slightly opalescent liquid to an opalescent liquid, but no particles were visible. Prescriptions that showed a large amount of precipitation were not further tested.

[0247] pH and concentration: Prescriptions F4 to F6 were all within the target values ​​with no significant changes.

[0248] SEC-HPLC: Observed at 40℃ for 4 weeks, the order of low molecular weight peaks was F6>F5>F4, and the order of high molecular weight peaks was F4>F5>F6.

[0249] CE-SDS(NR): Observed at 40℃ for 4 weeks, the purity of the CE(NR) main peak F6>F4, F5; the quality of the low molecular weight peak: F6>F4, F5.

[0250] CE-SDS(R): Observed at 40℃ for 4 weeks, the main peak (light chain and heavy chain) contents were ranked as follows: F6, F5>F4.

[0251] iCIEF: After 4 weeks at 40℃, the main peak showed a significant decrease.

[0252] DLS: Observed at 40℃ for 4 weeks, the particle size ranking is: F4>F5>F6.

[0253] Binding activity: After 4 weeks at 40℃, F4, F5 and F6 showed a significant decrease.

[0254] In summary, after 4 weeks of buffer system screening, formulations F4 to F6 (histidine buffer system) were significantly superior to the other formulations. The three formulations of the histidine buffer system with different pH values ​​had their own advantages and disadvantages in various indicators. Therefore, the histidine buffer system was determined to be the optimal buffer system. There is no optimal pH value yet, and all the experimental pH values ​​can be used to prepare antibody preparations. The formulation with the intermediate pH value (F5) was tentatively selected for the next step of excipient screening research. The histidine buffer system will be further studied in subsequent studies.

[0255] Example 3. Screening of excipients

[0256] 3.1 Excipient screening plan

[0257] Based on the buffer system screening results, four formulations (glycine, sucrose, trehalose, and mannitol) were added to the F5 formulation. The protein concentration was 10 mg / mL, and all formulations used 0.04% polysorbate 80 as a surfactant. Samples were subjected to accelerated testing at 40°C and freeze-thaw tests. The stability of each formulation was evaluated using appearance, pH, concentration, osmolarity, purity (by size exclusion chromatography (SEC-HPLC), capillary electrophoresis with non-reduced sodium dodecyl sulfate (CE-SDS(NR) and reduced sodium dodecyl sulfate (CE-SDS(R))), charge isomerism (by whole-column capillary isoelectric focusing (iCIEF)), dynamic light scattering (DLS), and binding activity (by ELISA) to identify the optimal excipient. The specific formulation composition and evaluation plan are shown in Table 7.

[0258] Table 7 Excipient screening scheme Note: X = appearance, pH, concentration, iCIEF, SEC-HPLC, CE-SDS (NR), CE-SDS (R), DLS, binding activity; Y = osmolarity; T0 = starting point of investigation; 1W = 1 week, and so on.

[0259] 3.2 Excipient screening results

[0260] The main results of excipient screening are summarized in Tables 8, 9, 10, 11, 12 and 13.

[0261] Table 8 Excipient screening 40℃ accelerated test results 1 Note: T0 = starting point of investigation; N / A = not applicable

[0262] Table 9 Excipient screening 40℃ accelerated test results 2 Note: T0 = survey starting point; ND = not detected

[0263] Table 10 Excipient screening 40℃ accelerated test results 3 Note: T0 = starting point of the survey

[0264] Table 11 Excipient screening freeze-thaw test results 1 Note: T0 = starting point of investigation; N / A = not applicable

[0265] Table 12 Excipient screening freeze-thaw test results 2 Note: T0 = survey starting point; ND = not detected

[0266] Table 13 Excipient screening freeze-thaw test results 3 Note: T0 = starting point of the survey

[0267] 3.3 Excipient screening conclusion

[0268] The sample concentration was 10 mg / mL, and five rounds of freeze-thaw tests were conducted at 40°C for 4 weeks and from -40°C to room temperature for excipient screening. Among them, the appearance of all prescriptions was a colorless slightly opalescent liquid with no visible particles, the pH was within the target value, and the concentration did not change significantly.

[0269] (1) Accelerated observation at 40°C for 4 weeks.

[0270] SEC-HPLC purity: No significant difference was found among the various formulations.

[0271] CE-SDS (NR): F5-2, F5-3, F5-4>F5-1. The results showed that the purity of the formulation containing glycine was slightly lower than that of other formulations.

[0272] CE-SDS(R): There were no significant differences among the formulations.

[0273] iCIEF: The degree of change in the acidic peaks F5-2 and F5-3 was slightly lower than that in other formulations, but the changes in the acidic and basic peaks did not lead to differences in binding activity.

[0274] Binding activity: All were reduced to between 40% and 60%, with no significant difference among the formulations.

[0275] (2) Freeze and thaw from -40℃ to room temperature for 5 cycles.

[0276] SEC-HPLC: F5-2, F5-3>F5-1, F5-4. The main peak purity of the formulations containing sucrose and trehalose was slightly higher than that of the formulation containing glycine and mannitol.

[0277] CE-SDS(NR), CE-SDS(R), iCIEF, binding activity: There were no significant differences among the formulations.

[0278] In summary, formulations F5-3 (containing trehalose) and F5-2 (containing sucrose) were superior to other formulations in the excipient screening process.

[0279] Since the binding activity in the liquid state has dropped to between 40% and 60%, the development of a freeze-dried preparation will be carried out.

[0280] In the freeze-dried powder state, the glass transition temperature (Tg) of the sample was detected using a differential scanning calorimeter (DSC). Under programmed temperature conditions, the temperature was raised from 0°C to 150°C. The relationship between the heat flow difference input to the sample and the reference during the heating process and the temperature was measured to detect the glass transition temperature of the freeze-dried powder sample. Generally, the higher the glass transition temperature of a substance, the better the stability of the substance at high temperature. Among them, the glass transition temperature (Tg) of F5-3 (containing trehalose) is 115.86°C, and the glass transition temperature (Tg) of F5-2 (containing sucrose) is 56.97°C. The glass transition temperature of F5-3 is significantly higher than that of the F5-2 prescription, indicating that trehalose can improve the stability of the preparation within a reasonable concentration range. Therefore, trehalose was determined to be the optimal excipient, and trehalose was used for the next step of surfactant screening.

[0281] Example 4. Surfactant screening

[0282] 4.1 Surfactant screening scheme

[0283] Surfactants at varying concentrations, including 0.02%, 0.04%, 0.06%, and 0.08% polysorbate 80 and poloxamer 188, were added to the screened buffer systems and excipients, resulting in eight formulations. Each formulation had a protein concentration of 10 mg / mL. The stability of each formulation was evaluated using an accelerated test at 40°C and shaking at 300 rpm at 25°C. Appearance, pH, concentration, surfactant concentration, purity (by size exclusion chromatography (SEC-HPLC), capillary electrophoresis with non-reduced sodium dodecyl sulfate (CE-SDS(NR) and reduced sodium dodecyl sulfate (CE-SDS(R))), charge isomerism (by whole-column capillary isoelectric focusing (iCIEF)), dynamic light scattering (DLS), insoluble particulate matter, and binding activity were used as indicators to screen the optimal surfactant. The specific formulation composition and evaluation plan are shown in Table 14.

[0284] Table 14 Surfactant screening scheme Note: X = appearance, pH, concentration, surfactant concentration, iCIEF, SEC-HPLC, CE-SDS (NR), CE-SDS (R), DLS, activity; Y = insoluble particles; T0 = starting point of investigation; 1W = 1 week; 1D = 1 day, and so on.

[0285] 4.2 Surfactant screening results

[0286] The main results of surfactant screening are summarized in Tables 15, 16, 17, 18, 19 and 20.

[0287] Table 15 Surfactant screening 40 ° C accelerated test results 1 Note: T0 = starting point of investigation; N / A = not applicable

[0288] Table 16 Surfactant screening 40 ° C accelerated test results 2 Note: T0 = survey starting point; ND = not detected

[0289] Table 17 Surfactant screening 40 ° C accelerated test results 3 Note: T0 = starting point of the survey

[0290] Table 18 Surfactant screening shaking test results 1 Note: T0 = starting point of investigation; N / A = not applicable

[0291] Table 19 Surfactant screening shaking test results 2 Note: T0 = survey starting point; ND = not detected

[0292] Table 20 Surfactant screening shaking test results 3 Note: T0 = starting point of the survey

[0293] 4.3 Conclusion of surfactant screening

[0294] Surfactant screening was performed using a 10 mg / mL sample concentration at 40°C for 4 weeks and at 25°C with shaking at 300 rpm for 7 days. Appearance, pH, and concentration: Formulation F5-3-8, shaken at 25°C and 300 rpm for 7 days, was colorless, slightly opalescent, and had visible feather-like particles. All other formulations and other investigation conditions were colorless, slightly opalescent liquids with no visible particles. The pH and concentration remained within target values, with no significant changes. There were no significant changes in insoluble particulates.

[0295] (1) After 4 weeks of observation at 40℃, the quality indicators of all prescriptions showed a downward trend.

[0296] SEC-HPLC: The formulation containing polysorbate 80 was slightly better than that containing poloxamer 188.

[0297] The downward trends of CE-SDS(NR), CE-SDS(R) and iCIEF were consistent.

[0298] Binding activity: The binding activity dropped below 50%.

[0299] (2) After shaking at 300 rpm at 25°C for 7 days, except for the appearance of the F5-3-8 formulation, the quality indicators of other formulations did not show a significant decrease.

[0300] From the above experimental results, it can be seen that polysorbate 80 and poloxamer 188 can both be used as surfactants in the formulation. The formulation containing polysorbate 80 is slightly better than the formulation containing poloxamer 188. Polysorbate 80 can stabilize the antibody preparation within a reasonable concentration range, and the concentration range of 0.02%-0.08% is its optimal concentration range. Furthermore, considering the production operational space and the principle of excipient use, 0.04% polysorbate 80 was selected as the surfactant for the next experiment.

[0301] Example 5. Determination of the content of each prescription component

[0302] 5.1 Buffer system component screening plan

[0303] Combined with the buffer system screening results, within the range of the histidine buffer system, the diffusion interaction parameter (kD) in the colloidal stability was used as an indicator to screen different components of the buffer system, including different contents and different pH values. If kD is a positive value, then in this buffer system, the protein molecules are mutually repelled, indicating that aggregation is not easily formed. The larger the positive value, the stronger the repulsion. If kD is a negative value, then in this buffer system, the protein molecules are mutually attracted, indicating that aggregation is easily formed. The larger the absolute value of the negative value, the stronger the attraction. The specific scheme is shown in Table 21.

[0304] Table 21 Buffer system component content and pH screening

[0305] 5.2 Buffer system component screening results

[0306] The main results of the buffer system component screening are summarized in Table 22.

[0307] Table 22 Buffer system component screening results

[0308] 5.3 Determination of buffer system components and the content of each prescription component

[0309] 5.3.1 Conclusion of buffer system component screening

[0310] Combined with the data of Example 2, it can be confirmed that in the histidine buffer system within the range of 5-20 nM, the stability of the composition meets the requirements and is not prone to forming aggregates.

[0311] Based on the buffer system screening results, we screened different buffer system component contents and pH values ​​within the histidine buffer system, using the diffusion interaction parameter (kD) of colloidal stability as an indicator. The results showed that the F4-B formulation had the highest kD. Within this buffer system component content and pH range, the mutual repulsion between protein molecules was the strongest, making aggregation least likely. Therefore, we ultimately selected the F4-B formulation (5mM histidine-histidine hydrochloride, pH 5.5) as the optimal buffer system formulation.

[0312] 5.3.2 Determination of the content of each prescription component

[0313] Based on adjustments to the buffer system components, and considering the convenience of industrial production and drug safety, the excipient (trehalose) content was adjusted to 9.5% (w / v), while the surfactant concentration remained unchanged. The final formulation was 5 mM histidine-histidine hydrochloride, 9.5% trehalose (w / v), and 0.04% polysorbate 80 (w / v), pH 5.5. As shown in Table 23, the osmotic pressure results of the final formulation were within the specification range of 290 ± 50 mOsmol / kg.

[0314] Example 6. Preparation of the final formulation of Tri-52 lyophilized preparation

[0315] The Tri-52 solution with a final protein concentration of 10 mg / mL was aseptically dispensed into vials, half-stoppered, and freeze-dried to obtain a Tri-52 lyophilized preparation. In this embodiment, a vacuum freeze dryer is used to freeze-dry the Tri-52 solution. The freeze-drying steps include: (1) cooling the drying box of the vacuum freeze dryer to -45°C and maintaining it for 3 to 5 hours; (2) raising the temperature of the drying box to -15°C and maintaining it for 3 to 5 hours; (3) cooling the drying box to -45°C and maintaining it for 3 to 5 hours. During the pre-freezing process, the heating and cooling rate is maintained at 0.5°C to 1°C / min; (4) the vacuum degree of the primary drying is 0.1mBar, and the temperature is maintained at -30 to -20°C. The total time of this step should be greater than or equal to 28 hours; (5) after the primary drying, the temperature is raised to 25°C at a heating rate of 0.1 to 0.3°C / min to start desorption drying, the vacuum degree is maintained at 0.1mBar, and it is maintained for 1 to 2 hours, and then the vacuum is applied to the limit, the temperature is kept unchanged, and the maintenance time is greater than or equal to 15 hours. Among them, steps (1) to (3) are pre-freezing, (4) are primary drying, and (5) are desorption drying.

[0316] Example 7. Stability test of final formulation

[0317] The Tri-52 lyophilized preparation was prepared using the preparation method of Example 6, and an accelerated stability test at 40°C was performed. The main indicators include appearance, reconstitution time, osmotic pressure molar concentration, pH, concentration, moisture, purity, charge isomerism, insoluble particles, activity, etc. Based on the results of the screening of the buffer system, excipients, surfactants and buffer system component content and pH, a prescription was finally determined: 10 mg / mL Tri-52, 5 mM histidine-histidine hydrochloride, 9.5% trehalose (w / v), 0.04% polysorbate 80 (w / v), pH 5.5, which was divided into vials, freeze-dried, and a sealed packaging system composed of a rubber stopper and an aluminum-plastic cap was used. A 4-week 40°C stability test was carried out.

[0318] The results are shown in Tables 23, 24, and 25, and Figures 2 to 5.

[0319] Table 23 Stability test results of final formulation-1 Note: T0 = starting point of investigation; N / A = not applicable

[0320] Table 24 Stability test results of final formulation-2 Note: T0 = starting point of the survey

[0321] Table 25 Stability test results of final formulation-3 Note: T0 = starting point of the survey

[0322] After 4 weeks of accelerated stability testing at 40°C, no significant changes were observed in appearance, reconstitution time, osmolarity, pH, concentration, SEC-HPLC, CE-SDS (NR), CE-SDS (R), insoluble particulate matter, or activity. Moisture content was ≤3%. A slight decrease in the main iCIEF peak was observed for charge isomerization, but this did not affect activity. This demonstrates that the protein in this formulation has excellent stability.

Claims

1. A composition comprising a multispecific antibody, wherein the composition is a lyophilized preparation, wherein the multispecific antibody comprises: (1) a first antigen-binding portion that binds to a tumor cell BCMA antigen; (2) a second antigen binding portion that binds to the tumor cell GPRC5D antigen; (3) A third antigen binding portion that binds to the CD3 antigen on immune cells.

2. The composition according to claim 1, wherein: (1) The first antigen-binding portion that binds to BCMA comprises HCDR1, HCDR2 and HCDR3 of VHH, wherein the HCDR1 comprises the sequence shown in SEQ ID NO.2, the HCDR2 comprises the sequence shown in SEQ ID NO.3, and the HCDR3 comprises the sequence shown in SEQ ID NO.4; (2) the second antigen-binding portion that binds to GPRC5D comprises a heavy chain variable region and a light chain variable region of an antibody or a fragment thereof, wherein the heavy chain variable region comprises HCDR1 of the sequence shown in SEQ ID NO.6, HCDR2 of the sequence shown in SEQ ID NO.7, and HCDR3 of the sequence shown in SEQ ID NO.8; and wherein the light chain variable region comprises LCDR1 of the sequence shown in SEQ ID NO.10, LCDR2 of the sequence shown in SEQ ID NO.11, and LCDR3 of the sequence shown in SEQ ID NO.12; and (3) The third antigen-binding portion that binds to CD3 comprises a heavy chain variable region and a light chain variable region of an antibody or a fragment thereof, wherein the heavy chain variable region comprises HCDR1 of SEQ ID NO.14, HCDR2 of SEQ ID NO.15, and HCDR3 of SEQ ID NO.16; and wherein the light chain variable region comprises LCDR1 of SEQ ID NO.18, LCDR2 of SEQ ID NO.19, and LCDR3 of SEQ ID NO.

20.

3. The composition according to claim 2, wherein: (1) The first antigen-binding portion that binds to BCMA comprises a VHH having a sequence as shown in SEQ ID NO.1; (2) the second antigen-binding portion that binds to GPRC5D comprises a heavy chain variable region of the sequence shown in SEQ ID NO.5 and a light chain variable region of the sequence shown in SEQ ID NO.9; and (3) The third antigen-binding portion that binds to CD3 comprises a heavy chain variable region with a sequence as shown in SEQ ID NO.13 and a light chain variable region with a sequence as shown in SEQ ID NO.

17.

4. The composition according to claim 2 or 3, wherein the multispecific antibody comprises the Fc domain of human IgG4. The composition according to claim 4 , wherein the human IgG4 Fc domain comprises different Fc1 and Fc2.

6. The composition according to claim 5, wherein the different Fc1 and Fc2 comprise amino acid mutations that alter effector function, wherein the amino acid mutations that alter effector function comprise S228P, F234A and / or L235A mutations of IgG4 Fc.

7. The composition according to claim 6, wherein the different Fc1 and Fc2 comprise amino acid mutations that promote heterodimerization, preferably, the amino acid mutations that promote heterodimerization comprise knob-into-hole (KIH) mutations; more preferably, the knob-into-hole mutations comprise: (a) either Fc region comprises a T366W mutation; and (b) the other Fc region comprises T366S, L368A and / or Y407V mutations.

8. The composition according to claim 7, wherein the multispecific antibody comprises a first heavy chain with a sequence as shown in SEQ ID NO.21, a second heavy chain with a sequence as shown in SEQ ID NO.22, a first light chain with a sequence as shown in SEQ ID NO.23, and a second light chain with a sequence as shown in SEQ ID NO.

23.

9. The composition according to claim 8, wherein any one of the Fc regions further comprises a His-tag, and the other Fc region further comprises a Flag-tag.

10. The composition of claim 1, wherein the multispecific antibody comprises a first light chain, a second light chain, a first heavy chain, and a second heavy chain, wherein: (1) The first light chain and the second light chain are identical and have the following structure: VL a -CL; and (2) The first heavy chain and the second heavy chain are different and have the following structures respectively: VHH b -L1-VH a -CH1-Fc1 and VH a -CH1-L2-VH CD3 -L3-VL CD3 -Fc2; optionally, one heavy chain also has a Flag tag at the carboxyl terminus and / or the other heavy chain also has a His tag at the carboxyl terminus; Among them, VH a and VL a The heavy chain variable region and light chain variable region that specifically bind to GPRC5D, VHH b is a nanobody that specifically binds to BCMA, VH CD3 and VL CD3 They are the heavy chain variable region and light chain variable region that specifically bind to CD3, and L1, L2, and L3 are the same or different linkers, respectively.

11. The composition according to claim 10, wherein The Fc1 and Fc2 of the multispecific antibody are different, Fc1 is knob-Fc and Fc2 is hole-Fc; or, Fc1 is hole-Fc and Fc2 is knob-Fc; preferably, the knob-Fc comprises a T366W mutation, and / or the hole-Fc comprises T366S, L368A and / or Y407V mutations; preferably, Fc1 and Fc2 are Fc regions of IgG4; more preferably, Fc1 is knob-Fc, and Fc2 is hole-Fc.

12. The composition according to claim 11, wherein the Fc1 and Fc2 of the multispecific antibody are different and have amino acid mutations that alter effector function; preferably, the amino acid mutations that alter effector function comprise S228P, F234A and / or L235A mutations of IgG4 Fc.

13. The composition according to any one of claims 1 to 12, wherein the composition further comprises a buffer system and a surfactant.

14. The composition according to claim 13, wherein the buffer system comprises a citrate buffer solution, a histidine buffer solution and / or a phosphate buffer solution.

15. The composition according to claim 14, wherein the buffer system comprises histidine-histidine hydrochloride buffer solution, and the content of the histidine-hydrochloric acid buffer solution is about 5-20 mM.

16. The composition according to claim 14, wherein the buffer system is a histidine-histidine hydrochloride buffer solution, and the content of the histidine-hydrochloric acid buffer solution is about 5 mM, about 10 mM or about 20 mM.

17. The composition of claim 13, wherein the surfactant comprises polysorbate 80 and / or poloxamer 188.

18. The composition according to claim 17, wherein the surfactant comprises polysorbate 80, and the content of polysorbate 80 is about 0.02-0.08% (w / v).

19. The composition of claim 17, wherein the surfactant is polysorbate 80, and the content of polysorbate 80 is about 0.2%, 0.4%, 0.6% or 0.8% (w / v).

20. The composition according to any one of claims 1 to 19, wherein the composition further comprises a stabilizer comprising glycine, sucrose, trehalose and / or mannitol.

21. The composition according to claim 20, wherein the stabilizer comprises trehalose, and the content of the trehalose is about 8.0-9.5% (w / v).

22. The composition of claim 21, wherein the stabilizer is trehalose, and the content of the trehalose is about 8%, about 8.5%, about 9% or about 9.5% (w / v).

23. The composition according to any one of claims 1 to 22, wherein the pH of the composition is about 5.0 to 7.0; preferably, the pH of the composition is about 5.5 to 6.0; more preferably, wherein the pH of the composition is about 5.

5.

24. A composition according to any one of claims 1 to 23, comprising: (1) Multispecific antibodies; (2) about 5 to 20 mM histidine-histidine hydrochloride buffer system; (3) about 0.02% to 0.08% (w / v) polysorbate 80; and (4) about 8.0% to 9.5% (w / v) trehalose; in, The pH of the composition is about 5.0 to 7.

5.

25. The composition according to claim 24, wherein The composition comprises: (1) Multispecific antibodies; (2) about 5 mM histidine-histidine hydrochloride buffer system; (3) about 0.04% (w / v) polysorbate 80; and (4) about 9.5% (w / v) trehalose; Wherein, the pH of the composition is about 5.

5.

26. The composition according to any one of claims 1 to 25, wherein The composition further comprises a solvent, which comprises sterile purified water and / or water for injection.

27. A method for preparing the composition of any one of claims 1 to 26, comprising the steps of: (1) fully mixing the buffer component, the surfactant and the stabilizer with a solvent to obtain a mixed solution; (2) thoroughly mixing the multispecific antibody molecule with the mixed solution; Optionally, the preparation method further includes the steps of sterilization and heat source removal.

28. A lyophilized powder preparation, wherein the lyophilized powder is obtained by freeze-drying the composition according to any one of claims 1 to 26; optionally, the composition is sterilized and pyrogen-free before freeze-drying.

29. The lyophilized powder formulation according to claim 28, wherein the lyophilized powder formulation is used for subcutaneous injection in a patient after reconstitution.

30. A method for preparing a lyophilized powder preparation, comprising freeze-drying the composition prepared according to the method of claim 27.

31. Use of the composition according to any one of claims 1 to 26 or the lyophilized powder preparation according to claim 28 or 29 in the preparation of a medicament for treating a tumor; preferably, the tumor is a B-cell lymphoma; more preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is relapsed / refractory multiple myeloma (R / RMM).

32. A composition according to any one of claims 1 to 26, or a lyophilized powder formulation according to claim 28 or 29, for treating a subject suffering from a tumor; preferably, the tumor is a B-cell lymphoma; more preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is relapsed / refractory multiple myeloma (R / RMM).

33. A method for treating a tumor in a subject, comprising administering a composition according to any one of claims 1 to 26, or a lyophilized powder formulation according to claim 28 or 29 to the subject, preferably, the tumor is a B-cell lymphoma; more preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is relapsed / refractory multiple myeloma (R / RMM).

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