Formulation of tri-specific antibody binding to human GPRC5d / BCMA / CD3 and use thereof

Abstract

Provided are a pharmaceutical formulation of a tri-specific antibody binding to human GPRC5D / BCMA / CD3 and a use thereof. The pharmaceutical formulation comprises a tri-specific antibody binding to human GPRC5D / BCMA / CD3, and further comprises a stabilizer, a buffer and / or a surfactant. The GPRC5D / BCMA / CD3 tri-specific antibody pharmaceutical formulation has good stability, can still meet pharmaceutical use requirements after long-term storage and exposure to strong light and high temperature, and has a wide application prospect.

Description

Trispecific antibody formulations combining human GPRC5D / BCMA / CD3 and their applications

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to Chinese patent application No. 202411053052.5, filed on August 1, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of antibody formulations, specifically relating to a pharmaceutical formulation of a trispecific antibody that binds to human GPRC5D / BCMA / CD3 and its uses. Technical Background

[0004] GPRC5D (G protein-coupled receptor, class C, group 5, member D) is a G protein-coupled receptor belonging to the GPCR family. GPRC5D is primarily expressed in tissues closely related to the immune system, such as bone marrow and thymus, while its expression in other tissues is relatively low. GPRC5D exhibits typical characteristics of the GPCR family, including seven α-helical transmembrane regions. The outer cytoplasmic side contains ligand-binding sites, while the inner cytoplasmic side interacts with G proteins. Upon ligand binding, GPRC5D activates G proteins, triggering intracellular signaling events and influencing cellular function. GPRC5D is specifically and highly expressed in multiple myeloma (MM) cells, and bispecific antibodies against GPRC5D, such as talquetamab and cevostamab, have shown efficacy in MM patients in clinical trials.

[0005] BCMA (B-cell maturation antigen, also known as TNFRSF17 or CD269) is a member of the tumor necrosis factor receptor (TNFR) superfamily. It is primarily expressed in mature B lymphocytes and plasma cells, and is virtually undetectable in other normal human cells. After binding to B-cell activating factor (BAFF) and the proliferation-inducing ligand (APRIL), BCMA activates the NF-κB and JNK signaling pathways, maintaining the survival, differentiation, and maturation of myeloma cells. BCMA is highly expressed in all MM cells, but almost not in other tissues. As an ideal target for MM treatment, several therapeutic studies have been conducted, including CAR-T cell therapy, bispecific T-cell binding antibodies (BiTE), and antibody-drug conjugates (ADCs). Some marketed products include Belantamab mafodotin (a BCMA-ADC product) and Abecma.

[0006] (BCMA-CAR-T product).

[0007] CD3 is a receptor on the surface of T cells, playing a crucial role in the immune response and activating T cells to exert tumor-killing effects. The antibody described in this article is a trispecific antibody targeting BCMA / GPRC5D / CD3, composed of an anti-CD3 binding domain, an anti-BCMA binding domain, and an anti-GPRC5D binding domain. This trispecific antibody performs dual antigen recognition on plasma cells, potentially enhancing binding to tumor cells through dual-target affinity, thereby effectively eliminating malignant clonal populations and preventing tumor antigen loss-mediated drug resistance.

[0008] Drug stability is one of the important indicators for ensuring drug efficacy and safety. In order to make the trispecific antibody targeting BCMA / GPRC5D / CD3 suitable for clinical application, it is necessary to conduct formulation development research on specific antibodies. Summary of the Invention

[0009] The inventors obtained a trispecific antibody that binds to human GPRC5D / BCMA / CD3 (see PCT / CN2024 / 076521 and PCT / CN2024 / 076517), and further explored and studied its formulation. Finally, they obtained a liquid or lyophilized formulation that is most suitable for this trispecific antibody and can stably preserve the trispecific antibody. This formulation can effectively prevent the aggregation, degradation, oxidation or denaturation of the trispecific antibody, thereby maintaining the biological activity of its effective components and making it suitable for clinical use.

[0010] One object of this disclosure is to provide a stable pharmaceutical formulation of a specific trispecific antibody or antigen-binding fragment thereof that binds to human GPRC5D / BCMA / CD3, and its use therein.

[0011] This disclosure provides a pharmaceutical formulation of a GPRC5D / BCMA / CD3 trispecific antibody or its antigen-binding fragment thereof, comprising a GPRC5D / BCMA / CD3 trispecific antibody or its antigen-binding fragment thereof, and a stabilizer and / or buffer.

[0012] In one embodiment, the GPRC5D / BCMA / CD3 trispecific antibody or its antigen-binding fragment comprises a heavy chain single-domain antibody (VHH) domain that specifically binds GPRC5D, an antigen-binding fragment Fab that specifically binds BCMA, and a single-chain antibody (scFv) domain that specifically binds CD3.

[0013] In one embodiment, the GPRC5D / BCMA / CD3 trispecific antibody or its antigen-binding fragment comprises a first polypeptide, a second polypeptide, and a third polypeptide.

[0014] In one embodiment, the first polypeptide comprises: (i) a heavy chain domain of an antigen-binding fragment Fab that specifically binds to BCMA, (ii) a single-chain antibody (scFv) domain that specifically binds to CD3, and (iii) a first Fc domain.

[0015] The second polypeptide contains: the light chain domain of the Fab antigen-binding fragment that specifically binds to BCMA;

[0016] The third polypeptide comprises: (i) a heavy chain single-domain antibody (VHH) domain that specifically binds to GPRC5D and (ii) a second Fc domain.

[0017] The heavy chain domain of the Fab antigen-binding fragment of the first polypeptide, which specifically binds to BCMA, and the light chain domain of the Fab antigen-binding fragment of the second polypeptide, which specifically binds to BCMA, form a binding site against BCMA; the single-chain antibody (scFv) domain that specifically binds to CD3 forms a binding site against CD3; the heavy chain single-domain antibody (VHH) domain that specifically binds to GPRC5D forms a binding site against GPRC5D; and the first Fc domain and the second Fc domain associate with each other.

[0018] The first Fc domain comprises a first CH2 domain and a first CH3 domain of immunoglobulin, wherein the C-terminus of the first CH2 domain is fused to the N-terminus of the first CH3 domain; and the second Fc domain comprises a second CH2 domain and a second CH3 domain of immunoglobulin, wherein the C-terminus of the second CH2 domain is fused to the N-terminus of the second CH3 domain.

[0019] Preferably, the first CH3 domain contains a "knob" structure, and the second CH3 domain contains a "hole" structure; more preferably, the "knob" structure contains amino acid substitutions of S354C and T366W, and the "hole" structure contains amino acid substitutions of Y349C, T366S, L368A, and Y407V.

[0020] Preferably, to reduce the ADCC activity of the antibody, the first and / or second Fc domains contain amino acid substitutions of L234A, L235A and / or G237A.

[0021] Preferably, the second Fc domain of the third polypeptide contains an amino acid substitution of H435R.

[0022] Preferably, the Fc domain is derived from IgG1.

[0023] The CD3-specific scFv domain comprises a heavy chain variable region and a light chain variable region. Preferably, the heavy chain variable region and the light chain variable region are connected by a first linker, wherein the C-terminus of the heavy chain variable region is fused to the N-terminus of the linker, and the C-terminus of the first linker is fused to the N-terminus of the light chain variable region. Preferably, the first linker comprises the amino acid sequence G4S.

[0024] In one embodiment, the N-terminus of the first Fc domain is fused to the C-terminus of the CD3-specific scFv domain. Preferably, the N-terminus of the first Fc domain is fused to the C-terminus of the CD3-specific scFv domain via a second linker. More preferably, the second linker comprises the amino acid sequence EPKSS.

[0025] In one embodiment, the heavy chain domain of the antigen-binding fragment Fab that specifically binds to BCMA is connected to the scFv domain that specifically binds to CD3 via a third linker, wherein the C-terminus of the heavy chain domain of the antigen-binding fragment Fab is fused to the N-terminus of the third linker, and the C-terminus of the third linker is fused to the N-terminus of the scFv domain that specifically binds to CD3. Preferably, the third linker comprises the amino acid sequence G4S.

[0026] In one embodiment, the C-terminus of the heavy chain single-domain antibody (VHH) domain that specifically binds to GPRC5D is fused to the N-terminus of the second Fc domain. Preferably, the C-terminus of the VHH domain is fused to the N-terminus of the second Fc domain via a fourth linker. More preferably, the fourth linker comprises the amino acid sequence EPKSS.

[0027] In one embodiment, the first polypeptide comprises the following structure: a heavy chain domain of the BCMA-specific antigen-binding fragment Fab - a third linker - a CD3-specific scFv domain - a second linker - a first Fc domain. Preferably, the first polypeptide comprises the following structure: a heavy chain variable region of the BCMA-specific antigen-binding fragment Fab - CH1 of the BCMA-specific antigen-binding fragment Fab - a third linker - a heavy chain variable region of the CD3-specific scFv domain - a first linker - a light chain variable region of the CD3-specific scFv domain - a second linker - a first CH2 - a first CH3.

[0028] In one embodiment, the second polypeptide comprises the following structure: a light chain variable region – a light chain constant region of the antigen-binding fragment Fab that specifically binds to BCMA.

[0029] In one embodiment, the third polypeptide comprises the following structure: a VHH domain that specifically binds to GPRC5D - a fourth linker - a second Fc domain. Preferably, the third polypeptide comprises the following structure: a VHH domain that specifically binds to GPRC5D - a fourth linker - a second CH2 - a second CH3.

[0030] Preferably, the CD3-specific scFv domain comprises HCDR1 as shown in SEQ ID NO: 21, HCDR2 as shown in SEQ ID NO: 22, HCDR3 as shown in SEQ ID NO: 23, LCDR1 as shown in SEQ ID NO: 24, LCDR2 as shown in SEQ ID NO: 25, and LCDR3 as shown in SEQ ID NO: 26. More preferably, the CD3-specific scFv domain comprises a heavy chain variable region as shown in SEQ ID NO: 19 and a light chain variable region as shown in SEQ ID NO: 20. More preferably, the scFv domain comprises the amino acid sequence shown in SEQ ID NO: 9.

[0031] Preferably, the BCMA-specific antigen-binding fragment Fab comprises HCDR1 as shown in SEQ ID NO: 10, HCDR2 as shown in SEQ ID NO: 11, and HCDR3 as shown in SEQ ID NO: 12, and / or LCDR1 as shown in SEQ ID NO: 13, LCDR2 as shown in SEQ ID NO: 14, and LCDR3 as shown in SEQ ID NO: 15. More preferably, the BCMA-specific antigen-binding fragment Fab comprises a heavy chain variable region as shown in SEQ ID NO: 6, and / or the BCMA-specific antigen-binding fragment Fab comprises a light chain variable region as shown in SEQ ID NO: 7. More preferably, the BCMA-specific antigen-binding fragment Fab comprises a heavy chain domain as shown in SEQ ID NO: 4, and / or the BCMA-specific antigen-binding fragment Fab comprises a light chain domain as shown in SEQ ID NO: 5.

[0032] Preferably, the heavy chain single-domain antibody (VHH) domain that specifically binds to GPRC5D comprises HCDR1 as shown in SEQ ID NO:16, HCDR2 as shown in SEQ ID NO:17, and HCDR3 as shown in SEQ ID NO:18; more preferably, the VHH domain that specifically binds to GPRC5D comprises the amino acid sequence shown in SEQ ID NO:8.

[0033] In one embodiment, the first polypeptide of the GPRC5D / BCMA / CD3 trispecific antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 1, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2.

[0034] In one embodiment, the pharmaceutical formulation comprises a buffer. Preferably, the buffer is selected from one or more of histidine buffers, citrate buffers, succinate buffers, acetate buffers, arginine buffers, and tartrate buffers, or is self-buffering; more preferably, the buffer is selected from histidine buffers or succinate buffers, or combinations thereof, or is self-buffering.

[0035] Preferably, the self-buffering is free of any buffering agent; preferably, the histidine buffer is a histidine-histidine hydrochloride buffer or a histidine-aspartic acid buffer, and the succinate buffer is a succinic acid-sodium hydroxide buffer or a succinic acid-sodium succinate buffer. Preferably, the buffer does not contain a phosphate buffer.

[0036] Preferably, the concentration of the buffer is from about 0 mM to about 30 mM, more preferably from about 0 mM to about 10 mM, and most preferably about 0 mM.

[0037] Preferably, the concentration of the trispecific antibody or its antigen-binding fragment in the pharmaceutical preparation is about 0.5 mg / mL to about 30 mg / mL, about 0.5 mg / mL to about 25 mg / mL, about 2 mg / mL to about 15 mg / mL, about 15 mg / mL to about 25 mg / mL, about 5 mg / mL to about 10 mg / mL, about 10 mg / mL to about 25 mg / mL, or about 20 mg / mL to 30 mg / mL, preferably about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, or about 25 mg / mL, and most preferably 25 mg / mL.

[0038] In one embodiment, the pharmaceutical formulation includes a stabilizer, preferably selected from one or more of sugars, polyols, sodium chloride, arginine hydrochloride, glycine, and methionine; preferably, the stabilizer is a sugar and / or a polyol, more preferably a combination of sugar and polyol.

[0039] Preferably, the sugar is selected from sucrose and trehalose; preferably, the polyol is selected from mannitol or sorbitol; the preferred stabilizer is trehalose and / or mannitol, more preferably a combination of trehalose and mannitol.

[0040] Preferably, the concentration of the sugar is about 20 mg / mL to about 60 mg / mL, and the concentration of the polyol is about 10 mg / mL to about 50 mg / mL; most preferably, the concentration of the sugar is about 40 mg / mL, and the concentration of the polyol is about 30 mg / mL. In one specific embodiment, the stabilizer is a combination of about 40 mg / mL trehalose and about 30 mg / mL mannitol.

[0041] In one embodiment, the pharmaceutical formulation comprises a surfactant, preferably a polysorbate and / or poloxamer 188, more preferably a polysorbate 80.

[0042] Preferably, the concentration of the surfactant is from about 0.1 mg / mL to about 1.0 mg / mL, more preferably from about 0.3 mg / mL to about 0.5 mg / mL, and more preferably from about 0.3 mg / mL.

[0043] Preferably, the pH of the pharmaceutical preparation is from about 4.0 to about 7.0, more preferably from about 4.0 to about 5.5, and even more preferably from about 4.3 to about 4.7. In one specific embodiment, the pH of the pharmaceutical preparation is about 4.5.

[0044] Preferably, the pharmaceutical preparation comprises any two or three of the following: about 40 mg / mL of trehalose, about 30 mg / mL of mannitol, and about 0.3 mg / mL of polysorbate 80.

[0045] In one embodiment, this disclosure also provides a lyophilized formulation containing a GPRC5D / BCMA / CD3 trispecific antibody or an antigen-binding fragment thereof, said lyophilized formulation being obtained by freeze-drying the pharmaceutical formulation described in this disclosure.

[0046] In one embodiment, this disclosure also provides a lyophilized formulation containing a GPRC5D / BCMA / CD3 trispecific antibody or an antigen-binding fragment thereof, wherein the lyophilized formulation, upon reconstitution, can form a pharmaceutical formulation as described in this disclosure.

[0047] This disclosure also provides a product comprising a container containing a pharmaceutical formulation as described in this disclosure, or a lyophilized formulation as described in this disclosure.

[0048] This disclosure also provides the use of the pharmaceutical preparations described herein in the preparation of a medicament for treating or inhibiting diseases or conditions associated with tumor cell proliferation or tumor cell metastasis, wherein the diseases or conditions are preferably tumors or cancers, and the tumors are selected from: lymphomas such as multiple myeloma, and metastatic tumors of the above-mentioned tumors.

[0049] This disclosure also provides the use of the pharmaceutical preparations described herein for treating or inhibiting diseases or conditions related to tumor cell proliferation or tumor cell metastasis, wherein the diseases or conditions are preferably tumors or cancers, and the tumors are selected from: lymphomas such as multiple myeloma, and metastatic cancers of the above tumors.

[0050] This disclosure also provides a method for treating or inhibiting diseases or conditions associated with tumor cell proliferation or tumor cell metastasis, comprising administering to a subject in need a pharmaceutical preparation or a lyophilized preparation of the present disclosure, preferably, the disease or condition being a tumor or cancer, more preferably, the tumor or cancer being selected from lymphomas such as multiple myeloma, and metastatic cancers of the aforementioned tumors.

[0051] The pharmaceutical preparation of the anti-human GPRC5D / BCMA / CD3 trispecific antibody or its antigen-binding fragment disclosed herein has at least one of the following beneficial effects: the preparation has good stability and can still meet the requirements for pharmaceutical use after long-term storage, strong light and high temperature, and has broad application prospects. Attached Figure Description

[0052] Figure 1: Schematic diagram of the structure of anti-human GPRC5D / BCMA / CD3 trispecific antibody.

[0053] Figure 2: Trend of pH effect on SEC purity.

[0054] Figure 3: Antibody stability at different buffer salt concentrations (SEC purity).

[0055] Figure 4: Antibody stability at different buffer salt concentrations (nr-CE purity).

[0056] Figure 5: Antibody stability at different buffer salt concentrations (iCIEF purity).

[0057] Figure 6: Antibody stability in different stabilizers (SEC purity).

[0058] Figure 7: Antibody stability in different stabilizers (nr-CE purity).

[0059] Figure 8: Antibody stability in different stabilizers (iCIEF purity).

[0060] Figure 9: Long-term stability of antibody preparations.

[0061] Figure 10: Accelerated stability of antibody preparations.

[0062] Figure 11: High-temperature stability of antibody preparations.

[0063] Figure 12: Photostability of antibody preparations. Detailed Implementation

[0064] the term

[0065] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0066] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0067] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0068] The three-letter and single-letter codes for amino acids used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0069] The term "antibody" in this article may include complete antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragment (i.e., antigen-binding part) or its single chain, and may also include products with antigen-specific binding ability formed by modifying complete antibodies or their antigen-binding fragments or their single chains (e.g., linking other peptides, rearranging functional units, etc.).

[0070] As used in this article, the term "antibody" typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies possess this structure. Each light chain contains a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH) and a constant domain.

[0071] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be assigned to one of two distinctly different types based on the amino acid sequence of its constant domain, termed κ and λ.

[0072] In the case of IgG, IgA, and IgD antibodies, this constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length, proline- and cysteine-rich segment. Each antibody class further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0073] The term "variable region" or "variable domain" indicates a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. Each variable region corresponding to the light chain / heavy chain forms an antibody binding site, resulting in a complete IgG antibody having two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more commonly, complementarity-determining regions (CDR). Each VH and VL has four backbone regions (FR), denoted as FR1, FR2, FR3, and FR4, respectively. Therefore, the CDR and FR sequences typically appear in the following sequence within the heavy chain variable domain (or light chain variable domain): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0074] It should be noted that the division of the CDR and FR in the variable region described in this disclosure is determined according to the Kabat definition. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any nomenclature system based on the monoclonal antibody sequence of this disclosure are explicitly kept within the scope of this disclosure.

[0075] The term "antibody fragment" includes at least a portion of a complete antibody. As used herein, a "fraction" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment in an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its immunogenicity-determining moiety, or a fusion protein product further derived from such fragment, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and trispecific antibodies or trispecific antibodies formed from antibody fragments, etc.

[0076] The term "Fab" or "Fab fragment" refers to a monovalent antibody fragment consisting of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain. The term "F(ab')2" or "F(ab')2 fragment" contains two Fab fragments and a hinge region, making it a bivalent antibody fragment.

[0077] The term "single-chain antibody" or "scFv" refers to a fusion protein comprising at least one antibody fragment including a variable region comprising a light chain and at least one antibody fragment including a variable region comprising a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0078] The “VHH domain,” also known as a heavy chain single-domain antibody, VHH, VHH antibody fragment, VHH antibody, or nanobody, is a variable domain of an antigen-binding immunoglobulin called a “heavy chain antibody” (i.e., an antibody lacking a light chain). The term “VHH domain” is used to distinguish this variable domain from the heavy chain variable domain (referred to herein as the “VH domain”) and light chain variable domain (referred to herein as the “VL domain”) present in conventional tetrapeptide chain antibody structures. The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (unlike the VH or VL domains in conventional tetrapeptide chain antibody structures, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain. The terms “heavy chain single-domain antibody,” “VHH domain,” “VHH,” “VHH domain,” “VHH antibody fragment,” “VHH antibody,” and “heavy chain antibody variable region” are used interchangeably. The “VHH domain” includes, but is not limited to, natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained through phage display technology.

[0079] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. As used herein, "amino acid substitution" or "replacement" means replacing an amino acid at a specific position in the parental polypeptide sequence with another amino acid. For example, substitution of S32A means that serine at position 32 is replaced by alanine.

[0080] The trispecific antibodies disclosed herein may also include substitutions or modifications to a constant region (e.g., Fc), including but not limited to amino acid residue substitutions, mutations, and / or modifications, which produce compounds having preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased yield, altered Fc ligand binding to an Fc receptor (FcR), enhanced or weakened ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity. In some aspects, antibody variants comprise an Fc region having one or more amino acid substitutions that weaken FcγR binding (e.g., substitutions at positions 234 and 235 of the Fc region). In one aspect, the substitutions are L234A and L235A.

[0081] The term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions. While the boundaries of the Fc region in the IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain; for example, the IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index.

[0082] The term "knob-into-Hole" refers to a modification used to promote the association of two polypeptide chains in a fungible cell (Fc), comprising a "knob" modification in one of the two polypeptide chains of Fc and a "hole" modification in the other. Generally, this method involves introducing a bulge ("knob") at the interface of the first polypeptide chain and a corresponding cavity ("hole") at the interface of the second polypeptide chain, such that the bulge can be placed within the cavity to promote heterodimer formation and inhibit homodimer formation. The bulge is constructed by replacing a small amino acid side chain from the interface of the first polypeptide chain with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity with the same or similar size as the bulge is created at the interface of the second polypeptide chain by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0083] Therefore, in one specific embodiment, in the CH3 domain of the first polypeptide chain of the Fc domain of the trispecific antibody of this disclosure, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a bulge within the CH3 domain of the first polypeptide chain, which can be placed in the cavity within the CH3 domain of the second polypeptide chain. Furthermore, in the CH3 domain of the second polypeptide chain of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second polypeptide chain, in which the bulge within the CH3 domain of the first polypeptide chain can be placed. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0084] The term "connector" refers to any tool used to connect two different functional units (e.g., antigen-binding fragments). Types of connectors include, but are not limited to, chemical connectors and peptide connectors. The sequence of peptide connectors is not limited. Peptide connectors are preferably non-immunogenic and flexible, such as those containing serine and glycine sequences. Depending on the specific construct, connectors can be long or short.

[0085] According to this disclosure, the linker connecting different functional units preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker comprises an amino acid sequence (G4S). n Or (G4S) n A, where n is any integer chosen from 1 to 10, preferably contains the amino acid sequence (G4S)3 or (G4S)3A. The linker connecting the VH and VL domains to form the scFv domain of VH-VL or VL-VH preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker comprises the amino acid sequence (G4S). n Or (G4S) n A, where n is any integer choice from 1 to 10, preferably containing the amino acid sequence (G4S)3 or (G4S).

[0086] As used herein, the broad category of "antibody" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, bispecific antibodies, trispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0087] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above.

[0088] The term "humanized antibody" refers to an antibody in which all or part of the amino acids, except for the CDR (Coefficient of Reduction) of a non-human antibody (such as a mouse antibody), have been replaced by corresponding amino acids derived from human immunoglobulins. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible, as long as they do not eliminate the antibody's ability to bind to a specific antigen. "Humanized" antibodies retain antigen specificity similar to the original antibody.

[0089] The term "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0090] The term "multispecific" refers to an antigen-binding molecule that can specifically bind to multiple different antigenic determinants. The term "antigen-binding molecule," in its broadest sense, refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and their derivatives, such as fragments. The terms "trispecific antigen-binding molecule," "trispecific binding molecule," or "trispecific antibody" refer to a binding molecule (e.g., an antibody or a molecule containing an antibody fragment) that specifically binds to three different antigens (or epitopes). The trispecific antibody of this disclosure binds to CD3, BAMA, and GPRC5D. The term "trispecific antibody" may also include prefixes such as "GPRC5D / BCMA / CD3," "GPRC5D / CD3 / BCMA," "CD3 / GPRC5D / BCMA," "CD3 / BCMA / GPRC5D," "BCMA / GPRC5D / CD3," or "BCMA / CD3 / GPRC5D" to indicate its specific binding to the three target proteins CD3, BCMA, and GPRC5D.

[0091] The term "specific binding" refers to a binding that is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antibody to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art.

[0092] When preparing antibodies, binding molecules, bispecific binding molecules or multispecific binding molecules using the variable regions of this disclosure, the constant regions are not particularly limited. Constant regions known to those skilled in the art or obtained independently can be used. Amino acid mutations (e.g., mutations that increase or decrease binding to Fc and receptor or FcRn) can also be introduced into the constant region.

[0093] There are no particular limitations on the methods used to obtain the binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules disclosed herein, and they can be obtained by any method. The binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules of the invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Culture media secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange.

[0094] As used in this article, the term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved through a variety of techniques known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0095] The term "stable transfection" or "stable transformation" refers to the introduction and integration of exogenous nucleic acids, DNA, or RNA into the genome of transfected cells. The term "stable transfectant" refers to a cell in which foreign DNA is stably integrated into the genomic DNA.

[0096] The term "biological activity" refers to the ability of an antibody to bind to an antigen and cause a measurable biological response, which can be measured in vitro or in vivo.

[0097] The terms "pharmaceutical formulation" or "formulation" refer to an article whose form of existence allows for the effective biological activity of the active ingredient and does not contain any other components that would be toxic to the subject to whom the formulation is to be administered.

[0098] The term "solution formulation" refers to a formulation that is liquid at a temperature of at least about 2°C to about 8°C under atmospheric pressure.

[0099] The term "lyophilized preparation" refers to a lyophilized preparation prepared using lyophilization techniques known in the art. Before administration to a patient, the lyophilized preparation should be reconstituted using an aqueous reconstitution component. This step allows the antibodies and other components in the lyophilized preparation to be redissolved to obtain a solution suitable for injection into the patient. The volume of the aqueous substance used for reconstitution determines the concentration of antibodies in the resulting pharmaceutical composition. Reconstitution with a smaller volume of aqueous substance than before lyophilization provides a more concentrated composition than before lyophilization. The reconstitution ratio (volume of the lyophilized preparation:volume of the preparation before lyophilization) can range from 1:0.5 to 1:6. The lyophilized preparations of this disclosure can be reconstituted to obtain aqueous compositions having concentrations of at least 5 mg / mL, 15 mg / mL, and 25 mg / mL of GPRC5D / BCMA / CD3 specific antibodies, with the volume of the reconstitution component selected accordingly. If necessary, the reconstituted preparation can be diluted before administration to a patient to deliver the intended dose as appropriate.

[0100] Typical reconstitution components for lyophilized formulations include sterile water or a buffer, optionally containing a preservative. If the lyophilized formulation includes a buffer, the reconstitution component may further include a buffer (which may be a buffer of the same or different lyophilized formulation), or it may not include a buffer (e.g., water for injection, physiological saline, or glucose injection).

[0101] The term "aggregation" refers to an antibody or fusion protein that has been found to aggregate with other antibody or fusion protein molecules, especially after freezing and / or agitation.

[0102] The term "stable" formulation refers to a formulation in which the protein substantially retains its physical and / or chemical stability and / or biological activity after storage. It exhibits stability when stored at refrigerated temperatures (2-8°C) for up to 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Furthermore, it demonstrates stability after storage at 25°C for periods including 1 month, 3 months, 6 months, or at 40°C for 30 days.

[0103] Typical acceptable standards for stability are as follows: Degradation of the active ingredient, typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the pharmaceutical formulation is a pale yellow, nearly colorless, clear liquid, or colorless or clear to slightly milky white, or a pale yellow, nearly colorless, clear liquid. The concentration and pH of the formulation vary by no more than ±10%. Truncation of no more than about 10%, preferably no more than 5%, is typically observed. Aggregation of no more than 10%, preferably no more than 5%, is typically formed.

[0104] The term "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical preparation. Suitable buffers are well known in the art and can be found in the literature. Preferred pharmaceutically acceptable buffers include, but are not limited to, histidine buffers, citrate buffers, succinate buffers, acetate buffers, arginine buffers, tartrate buffers, or mixtures thereof. The pH of the buffer can be adjusted using an acid or base known in the art to a value in the range of about 4.0 to about 6.0, particularly to a value in the range of about 4.0 to about 5.0, and most particularly to a pH of about 4.5.

[0105] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application. Stabilizers include, but are not limited to, sugars, amino acids, polyols, cyclodextrins, etc.

[0106] The term "surfactant" refers to a pharmaceutically acceptable excipient used to protect protein formulations against physical stresses such as agitation and shearing. Pharmaceutically acceptable surfactants include: polyoxyethylene dehydrated sorbitol fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g., in the trademark Brij...), and more. TM Those sold below) and polyoxyethylene-polyoxypropylene copolymers (poloxamer, Pluronic). Polyoxyethylene dehydrated sorbitol-fatty acid esters include polysorbate 20 (in the trademark Tween 20) TM(Sold below) and polysorbate 80 (under the trademark Tween 80) TM (Sales below).

[0107] The term "effective dose" refers to a dosage of the pharmaceutical formulation of the antibody or fragment of this disclosure that, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective dose can be readily determined by an attending physician skilled in the art by considering a variety of factors, such as: racial differences; weight, age, and health status; the specific disease involved; the severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0108] As used herein, the terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. Individuals disclosed herein include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0109] As used herein, the terms “disease,” “symptom,” or “disorder,” etc., refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. For example, “disease” includes, but is not limited to: tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or deficiencies in immune tolerance (such as transplant rejection).

[0110] As used in this article, the term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion into other tissues or organs. Tumor growth is typically uncontrolled and progressive, neither inducing nor inhibiting the proliferation of normal cells.

[0111] As used in this article, the term "treatment" refers to a clinical intervention in the process of attempting to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in the clinicopathological process. Treatment effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0112] The terms “G protein-coupled receptor C5 family subtype D” and “GPRC5D” specifically include the human GPRC5D protein, and include variants, subtypes, homologs and analogs of human GPRC5D that share at least one common epitope with GPRC5D (e.g., human GPRC5D), as exemplary human GPRC5D sequences can be found in GenBank accession number BC069341, NCBI reference sequence: NP_061124.1 and UniProtKB / Swiss-Prot accession number Q9NZD1 (see also Brauner-Osborne, H et al., 2001, Biochim. Biophys. Acta 1518, 237-248).

[0113] The term "BCMA" refers to the tumor-associated antigen B-cell maturation antigen, also known as TNFRSF17. An exemplary human BCMA sequence includes the human BCMA protein under accession number UniProt Q02223.

[0114] Example

[0115] The present disclosure is further described in detail through the following examples. Any changes to the concentration of the formulation components or the addition of other substances based on the present disclosure, without significantly affecting the stability of the trispecific antibody, are still considered part of the present disclosure.

[0116] Size exclusion chromatography (SEC)

[0117] Size exclusion chromatography is used to quantify polymers, monomers, and fragments. This assay utilizes... BEH SEC Column, A 3.5 μm, 7.8 x 300 mm column was used and run on a Waters e2695-2489 HPLC or Arc HPLC system. The mobile phase was 100 mM phosphate, 150 mM sodium chloride buffer, pH 6.8. A sample volume of 50 μg protein was injected, and the protein was eluted isocratically at a flow rate of 0.5 mL / min for 30 min. The absorbance of the eluent was measured at 280 nm. Integration was performed using Empower 3 software.

[0118] Capillary electrophoresis (CE-SDS)

[0119] The purity of the main peak was determined by non-reducing CE-SDS (nrCE). This determination was performed on a BECKMAN COULTER PA800plus capillary electrophoresis system using a 50 μm ID uncoated quartz capillary with an effective capillary separation length of 20 cm (total length 30.2 cm) and a PDA with a 220 nm bandwidth and a 10 nm detection bandwidth.

[0120] Charge heterogeneity (iCIEF)

[0121] The charge heterogeneity and isoelectric point of the sample were detected using full-column imaging capillary isoelectric focusing electrophoresis. The capillary was a 100 μm inner diameter FC-coated fused silica capillary with an effective separation length of 5 cm. During sample preparation, GE pharmalyte 3-10, GE pharmalyte 5-8, hydroxypropyl methylcellulose, and 4 mol / L urea were added to a final concentration of 2 mg / ml. The focusing voltage and time were 1.5 kV for 1 min and 3 kV for 9 min. The pI value of the target peak was calculated using the marker pI value.

[0122] The following specific embodiments are provided to illustrate this disclosure. It should be understood that these examples are merely illustrative and not intended to limit the scope of this disclosure.

[0123] Example 1: Structure and sequence information of GPRC5D / BCMA / CD3 trispecific antibody

[0124] The heavy chain domain sequence of the antigen-binding fragment Fab that binds to BCMA is shown in SEQ ID NO:4, and the light chain domain sequence is shown in SEQ ID NO:5.

[0125] The sequence of the VHH domain combined with GPRC5D is shown in SEQ ID NO:8.

[0126] The heavy chain variable region sequence of the CD3-binding single-chain antibody (scFv) domain is shown in SEQ ID NO:19, and the light chain variable region sequence is shown in SEQ ID NO:20. The heavy and light chain variable regions are connected by a flexible linker, with the structure: VH-(G4S)3-VL, as shown in SEQ ID NO:9. The scFv is then fused to the C-terminus of the Fab heavy chain domain of the BCMA antigen-binding fragment via the flexible linker.

[0127] The scFv domain binding to CD3 is fused to the C-terminus of the CH1 of the Fab heavy chain domain of the antigen-binding fragment binding to BCMA to form one arm of the trispecific antibody. At the same time, the VHH domain binding to GPRC5D and the Fc part with the "node" and "hole" structures are introduced to form the trispecific antibody binding GPRC5D / BCMA / CD3 disclosed herein (the structure and sequence are shown in Tables 1 and 2). The sequences of the three heterologous peptide chains constituting the trispecific antibody are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. A schematic diagram is shown in Figure 1.

[0128] To reduce the ADCC activity of the antibodies, the Fc domains of the final trispecific antibodies underwent amino acid substitutions at L234A, L235A, and G237A. The Fc domains in peptide chains containing scFv were designed as "knob" structures, including amino acid substitutions at S354C and T366W. The Fc domains in peptide chains without scFv were designed as "hole" structures, including amino acid substitutions at Y349C, T366S, L368A, and Y407V. Furthermore, to facilitate the purification of the trispecific antibodies, the heavy chain of the "hole" structure underwent H435R substitution.

[0129] Table 1. Structural description of trispecific antibodies

[0130] Table 2. Amino acid sequences of trispecific antibodies

[0131] Example 2: Preparation of GPRC5D / BCMA / CD3 trispecific antibody

[0132] The GPRC5D / BCMA / CD3 trispecific antibody was expressed by CHO cells. The cell fermentation broth was purified by disc centrifugation, deep filtration, and sterile filtration. The purification steps included affinity chromatography and low-pH virus inactivation, cation chromatography, UF / DF chromatography, removal of protein aggregates, debris, HCP, DNA, ProA, and other impurities, ultimately yielding the antibody stock solution.

[0133] Example 3 pH Screening Experiment

[0134] This embodiment investigates the effect of different pH conditions on the stability of the purified GPRC5D / BCMA / CD3 trispecific antibody from Example 2, in order to obtain an optimal pH range. Sucrose was used as a stabilizer at a concentration of 90 mg / mL, polysorbate 80 was selected as a surfactant at a concentration of 0.5 mg / mL, and succinic acid-sodium succinate was used as a buffer solution at a concentration of 10 mM. Specific formulation information is shown in Table 3.

[0135] Table 3 pH Screening Experiment Prescription Information Table

[0136] Protein concentration, SEC, nrCE, and iCIEF were analyzed in the above formulation. The experimental results are shown in Table 4.

[0137] Table 4 Results of pH screening experiment

[0138] The protein content of samples with different pH values ​​did not change significantly after being placed at 40℃±2℃ for one week. SEC-HPLC results showed that the SEC purity at day 0 decreased with increasing pH (see Figure 2); after one week of high-temperature treatment at 40℃, the purity of each formulation decreased slightly; nrCE-SDS results showed that after one week of high-temperature treatment, all formulations showed an increase in fragmentation, with the R1 formulation showing the largest increase, while the other groups showed little difference. iCIEF results showed that after one week of high-temperature treatment, the main peak of each formulation decreased, with the R4 formulation showing the most significant change of 4%, while the other groups showed little difference.

[0139] In summary, the pH screening experiment results indicate that a pH range of 4.0-5.0 is suitable to balance protein purity and stability, with pH 4.5 being the preferred value.

[0140] Example 4: Buffer Concentration Screening

[0141] The pH screening experiment results show that antibody purity is greatly affected by the formulation. In this example, the effect of buffer concentration on antibody purity and stability was mainly investigated. Specific formulation information is shown in Table 5.

[0142] Table 5. Buffer Concentration Screening Prescription Information

[0143] Note: / indicates that it does not contain buffer, i.e., it is self-buffered.

[0144] Protein concentration, SEC, nrCE, and iCIEF were analyzed in the above formulation. The experimental results are shown in Table 6.

[0145] Table 6 Results of buffer concentration screening experiment

[0146] Note: NA indicates not detected.

[0147] The results showed that after one week of storage at 40℃, the protein content of formulations R1, R2, and R3 did not change significantly, while the purity of SEC, nr-CE, and iCIEF decreased slightly, with little difference among the formulations. Different buffer concentrations mainly affected SEC purity; the lower the buffer concentration, the higher the SEC purity. After four weeks of storage at 40℃, the protein content of formulations R4 and R5 did not change significantly, while the purity of SEC, nr-CE, and iCIEF all decreased. Formulation R5 was superior to formulation R4, and the trends are shown in Figure 3-5.

[0148] In summary, the results of the buffer concentration screening experiment show that the formulation without buffers is slightly better than the formulation with buffers in terms of SEC purity and stability. Therefore, it is more appropriate to choose a self-buffering system without buffers.

[0149] Example 5: Screening of Stabilizer Types and Concentrations

[0150] Based on the self-buffered system and pH 4.5, the types and concentrations of stabilizers were screened. Specific formulation information is shown in Table 7.

[0151] Table 7. Information on Stabilizer Types and Concentrations for Prescription Screening

[0152] Protein concentration, SEC, nrCE, iCIEF, and osmolarity were analyzed for the above formulation. The experimental results are shown in Table 8.

[0153] Table 8 Results of the screening experiment for stabilizer types and concentrations

[0154] The results showed that after 4 weeks of storage at 40℃, the protein content of the samples did not change significantly, while the purity of SEC, nr-CE, and iCIEF all decreased, as shown in Figures 6-8. The stability of the various formulations was not significantly different. The isotonic range of the injectable formulation was 285–315 mOsm / kg, with the R4 formulation having an osmotic pressure closer to isotonic conditions. Considering stability, osmotic pressure, and cost, the preferred stabilizer was 4% (40 mg / ml) trehalose + 3% (30 mg / ml) mannitol.

[0155] Example 6: Screening of Surfactant Types and Doses

[0156] The type and concentration of surfactants can affect the formation of protein solution particles, thereby affecting protein stability. In this example, the types and concentrations of surfactants such as polysorbate 80, polysorbate 20, and poloxamer 188 were investigated, and the formulation design is shown in Table 9.

[0157] Table 9. Surfactant Types and Dosage Information for Prescription Screening

[0158] Protein concentration, SEC, and insoluble microparticles were analyzed in the above formulation. The experimental results are shown in Table 10.

[0159] Table 10 Results of the screening experiment for surfactant types and dosages

[0160] Note: NA indicates that the sampling point was not detected.

[0161] The results showed that after three freeze-thaw cycles and three days of shaking, no significant changes or differences were observed in protein content, SEC purity, and insoluble particulate matter among different surfactant formulations. Among formulations with different polysorbate 80 concentrations, except for formulation R1 which showed a slight increase in insoluble particulate matter after shaking, the other formulations remained stable in terms of protein content, SEC purity, and insoluble particulate matter after freeze-thaw cycles and shaking, with no significant differences among the formulations. Considering that polysorbate 20 is more easily degraded, has a stronger hemolytic effect, and is widely used, polysorbate 80 is the preferred surfactant, with a preferred concentration of 0.03% (i.e., 0.3 mg / ml).

[0162] Example 7 Stability Test

[0163] Based on the results of the above screening experiments, stability studies were conducted on the following drug formulation: 25 mg / ml anti-GPRC5D / BCMA / CD3 trispecific antibody, 40 mg / ml trehalose, 30 mg / ml mannitol, 0.3 mg / ml polysorbate 80, pH 4.5. The studies included: forced-condition experiments (high-temperature experiment, strong light irradiation experiment), accelerated experiments, and long-term experiments. The dosage form was selected as lyophilized. The lyophilization cycle parameters and testing conditions for the stability study samples are shown in Tables 11 and 12, respectively.

[0164] Table 11 Freeze-drying Cycle Parameters for Formulation Process

[0165] Table 12 Conditions for Formulation Stability Study

[0166] The results showed that the purity of SEC, non-reduced CE-SDS, and iCIEF remained stable during the 6-month observation period under long-term (5℃) and accelerated (25℃) conditions. Under high-temperature conditions (40℃), the purity of SEC and iCIEF decreased slightly, while other quality indicators showed no significant changes. Under light irradiation, the purity of iCIEF decreased significantly, and the purity of nrCE-SDS decreased slightly, while other quality indicators showed no significant changes. The trends are shown in Figure 9-12. These results indicate that the formulation can be stored long-term under the proposed storage conditions of 5℃±3℃ (2~8℃), protected from light, and during transportation.

[0167] The embodiments described above are merely exemplary and should not be construed as limiting the implementation of this disclosure. Any person skilled in the art will recognize or be able to identify numerous equivalents of specific compounds, materials, and operations without the need for unconventional experimentation. All such equivalents are within the scope of this disclosure and are included by the claims.

[0168] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent or patent application had been specifically and individually incorporated herein by reference.

Claims

1. A pharmaceutical preparation comprising a GPRC5D / BCMA / CD3 trispecific antibody or an antigen-binding fragment thereof, and a buffer and / or a stabilizer, the GPRC5D / BCMA / CD3 trispecific antibody or an antigen-binding fragment thereof comprising a heavy chain single-domain antibody (VHH) domain specifically binding to GPRC5D, an antigen-binding fragment Fab specifically binding to BCMA, and a scFv domain specifically binding to CD3; wherein the heavy chain single-domain antibody (VHH) domain specifically binding to GPRC5D comprises a HCDR1 as set forth in SEQ ID NO: 16, a HCDR2 as set forth in SEQ ID NO: 17, and a HCDR3 as set forth in SEQ ID NO: 18, the antigen-binding fragment Fab specifically binding to BCMA comprises a HCDR1 as set forth in SEQ ID NO: 10, a HCDR2 as set forth in SEQ ID NO: 11, a HCDR3 as set forth in SEQ ID NO: 12, a LCDR1 as set forth in SEQ ID NO: 13, a LCDR2 as set forth in SEQ ID NO: 14, and a LCDR3 as set forth in SEQ ID NO: 15, and the scFv domain specifically binding to CD3 comprises a HCDR1 as set forth in SEQ ID NO: 21, a HCDR2 as set forth in SEQ ID NO: 22, a HCDR3 as set forth in SEQ ID NO: 23, a LCDR1 as set forth in SEQ ID NO: 24, a LCDR2 as set forth in SEQ ID NO: 25, and a LCDR3 as set forth in SEQ ID NO:

26.

2. The pharmaceutical preparation of claim 1, wherein the VHH domain specifically binding to GPRC5D comprises an amino acid sequence as set forth in SEQ ID NO: 8, and / or, the antigen-binding fragment Fab specifically binding to BCMA comprises a heavy chain variable region as set forth in SEQ ID NO: 6, and / or, the antigen-binding fragment Fab comprises a light chain variable region as set forth in SEQ ID NO: 7, and / or the scFv domain specifically binding to CD3 comprises a heavy chain variable region as set forth in SEQ ID NO: 19 and a light chain variable region as set forth in SEQ ID NO:

20.

3. The pharmaceutical preparation of claim 2, wherein the antigen-binding fragment Fab specifically binding to BCMA comprises a heavy chain domain as set forth in SEQ ID NO: 4, and the antigen-binding fragment Fab specifically binding to BCMA comprises a light chain domain as set forth in SEQ ID NO: 5, and / or, the scFv domain comprises an amino acid sequence as set forth in SEQ ID NO:

9.

4. The pharmaceutical preparation of any one of claims 1-3, wherein the GPRC5D / BCMA / CD3 trispecific antibody or an antigen-binding fragment thereof comprises a first polypeptide, a second polypeptide, and a third polypeptide.

5. The pharmaceutical formulation of claim 4, wherein, the first polypeptide comprises: (i) a heavy chain domain of an antigen binding fragment Fab that specifically binds to BCMA, (ii) a single chain antibody (scFv) domain that is capable of specifically binding to CD3, and (iii) a first Fc domain; the second polypeptide comprises: a light chain domain of an antigen binding fragment Fab that specifically binds to BCMA; the third polypeptide comprises: (i) a heavy single domain antibody (VHH) domain that specifically binds to GPRC5D and (ii) a second Fc domain.

6. The pharmaceutical formulation of claim 4 or 5, wherein the first polypeptide of the GPRC5D / BCMA / CD3 trispecific antibody or antigen binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 1, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3, and the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

2.

7. The pharmaceutical formulation of any one of claims 1-6, further comprising a surfactant.

8. The pharmaceutical formulation of any one of claims 1-7, wherein the pharmaceutical formulation has a pH of about 4.0 to about 5.0, preferably, the pH of the pharmaceutical formulation is about 4.

5.

9. The pharmaceutical formulation of any one of claims 1-8, wherein the concentration of the GPRC5D / BCMA / CD3 trispecific antibody or antigen binding fragment thereof is about 0.5 mg / mL to about 30 mg / mL.

10. The pharmaceutical formulation of any one of claims 1-9, wherein the concentration of the GPRC5D / BCMA / CD3 trispecific antibody or antigen binding fragment thereof is 0.5 mg / mL to about 25 mg / mL or about 2 mg / mL to about 25 mg / mL.

11. The pharmaceutical formulation of any one of claims 1-10, wherein the concentration of the GPRC5D / BCMA / CD3 trispecific antibody or antigen binding fragment thereof is about 2 mg / mL to about 15 mg / mL, about 15 mg / mL to about 25 mg / mL, about 5 mg / mL to about 10 mg / mL, about 10 mg / mL to about 25 mg / mL, or about 20 mg / mL to 30 mg / mL.

12. The pharmaceutical formulation of any one of claims 1-9, wherein the concentration of the GPRC5D / BCMA / CD3 trispecific antibody or antigen binding fragment thereof is about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, or about 25 mg / mL.

13. The pharmaceutical formulation of any one of claims 1-12, wherein the buffer is one or several of a histidine salt buffer, an acetate buffer, a succinate buffer, an arginine buffer, a tartrate buffer, a citrate buffer, or self-buffering.

14. The pharmaceutical formulation of any one of claims 1-13, wherein the concentration of the buffer is about 0 mM to about 20 mM.

15. The pharmaceutical formulation of any one of claims 1-14, wherein the stabilizer is selected from one or more of sucrose, trehalose, mannitol, sorbitol.

16. The pharmaceutical formulation of claim 15, wherein the concentration of sucrose or trehalose is about 20 mg / mL to about 60 mg / mL.

17. The pharmaceutical formulation of any one of claims 15-16, wherein the concentration of mannitol or sorbitol is about 10 mg / mL to about 50 mg / mL.

18. The pharmaceutical formulation of any one of claims 15-17, wherein the stabilizer comprises about 40 mg / mL trehalose and about 30 mg / mL mannitol.

19. The pharmaceutical formulation of any one of claims 7-18, wherein the surfactant comprises polysorbate and / or poloxamer 188, preferably the concentration of polysorbate or poloxamer 188 is about 0.2 mg / mL to about 1 mg / mL.

20. The pharmaceutical formulation of claim 19, wherein the polysorbate is polysorbate 80 at a concentration of about 0.3 mg / mL.

21. The pharmaceutical formulation of any one of claims 1-20, which is a liquid pharmaceutical formulation.

22. A lyophilized formulation comprising a GPRC5D / BCMA / CD3 trispecific antibody or an antigen-binding fragment thereof, which is obtained by lyophilizing the pharmaceutical formulation of any one of claims 1-21.

23. An article of manufacture comprising a container comprising the pharmaceutical formulation of any one of claims 1-21, or the lyophilized formulation of claim 22.

24. Use of the pharmaceutical formulation of any one of claims 1-21 or the lyophilized formulation of claim 22 in the manufacture of a medicament for, or of a method of treating or inhibiting, a disease or disorder associated with tumor cell proliferation or tumor cell metastasis, preferably the disease or disorder is a tumor or cancer, more preferably the tumor or cancer is selected from lymphoma such as multiple myeloma, and metastatic cancer of the aforementioned tumors.

25. A method for treating or inhibiting a disease or disorder associated with tumor cell proliferation or tumor cell metastasis, comprising administering to a subject in need thereof the pharmaceutical formulation of any one of claims 1-21 or the lyophilized formulation of claim 22, preferably the disease or disorder is a tumor or cancer, more preferably the tumor or cancer is selected from lymphoma such as multiple myeloma, and metastatic cancer of the aforementioned tumors.

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