An antibody-drug conjugate and medical use thereof

Abstract

This invention relates to an anti-BCMA antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound, and its pharmaceutical uses. Specifically, the antibody-drug conjugate of this invention is composed of an anti-BCMA antibody or its antigen-binding fragment and an eczemab derivative linked by a linker. The antibody-drug conjugate of this invention or its pharmaceutically acceptable salt or solvent compound exhibits significant antitumor efficacy and good safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically, this invention relates to anti-BCMA antibody-drug conjugates and their pharmaceutical uses. Prior Technology

[0002] B cells are a type of lymphocyte that play a crucial role in adaptive humoral immunity and the production of antibodies that specifically recognize antigens. There are three subclasses of B cells: naive B cells, memory B cells, and plasma cells. During VDJ recombination, two or three segments of DNA are selected from a genomic library, and recombination can produce a combined array of antibody variable domains. Through further alteration of the variable domains encoded by different B cell lineages, up to 109 unique B cell lineages can be generated, each producing antibodies with unique target specificity. Many diseases involve B cells; malignant transformation of B cells leads to cancers, including some lymphomas such as multiple myeloma and Hodgkin's lymphoma. Autoimmune diseases also involve B cells, including systemic lupus erythematosus (SLE) and IgA nephropathy. Cancers and autoimmune diseases involving B cells can be considered as B cell dysfunction; therefore, a possible strategy for controlling these diseases is to use antibodies that target pathological B cells.

[0003] BCMA is a member of the TNF receptor superfamily and is a non-glycosylated intrinsic membrane receptor for its ligands BAFF (B cell activating factor) and APRIL (proliferation-inducing ligand). BCMA and its corresponding ligands play different roles in regulating humoral immunity, B cell development, and homeostasis. BCMA is expressed by amygdala memory B cells and germinal center B cells, and can be detected in the spleen, lymph nodes, thymus, adrenal glands, and liver. Analysis of various B cell lines shows that BCMA expression levels increase after maturation. Summary of the Invention

[0004] The purpose of this invention is to provide an antibody-drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvent compound thereof.

[0005]

[0006] in,

[0007] W is selected from -(CReRf)g-X1-(CReRf)u-X2-(CReRf)h-,

[0008] Re or Rf is independently selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl, or hydroxyalkyl; preferably, Re or Rf is independently selected from hydrogen or deuterium, more preferably hydrogen.

[0009] X1 or X2 is independently selected from N, H, O or S; preferably, X1 or X2 is independently selected from S; more preferably, it is O.

[0010] g, u, or h are each independently selected from 1, 2, 3, or 4; preferably, g, u, or h are each independently selected from 1, 2, or 3; more preferably, g is selected from 2.

[0011] y is 1~20; preferably 4~10; even better 4, 6, 8 or 10;

[0012] Ab represents anti-BCMA antibody or its antigen-binding fragment.

[0013] In a further preferred embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4 and HCDR3 shown in SEQ ID NO:5, and the light chain variable region comprises the antibody light chain variable region comprising LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:8.

[0014] In a further preferred embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0015] In a further preferred embodiment of the invention, the anti-BCMA antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

[0016] Preferably, the anti-BCMA antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, or IgG4;

[0017] More preferably, the anti-BCMA antibody or its antigen-binding fragment further comprises an IgG1 heavy chain constant region that exhibits enhanced ADCC toxicity following amino acid mutation;

[0018] Alternatively, the anti-BCMA antibody or its antigen-binding fragment may further include a heavy chain constant region as shown in SEQ ID NO:22.

[0019] In a further preferred embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region of a human antibody κ chain, λ chain or a variant thereof; more preferably, the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region of a human antibody κ chain; even more preferably, the anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region as shown in SEQ ID NO:23.

[0020] In a further preferred embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the sequences shown below: SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11;

[0021] And / or, the anti-BCMA antibody or its antigen-binding fragment is selected from the light chain variable region shown in the following sequence, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

[0022] In a further preferred embodiment of the present invention, the anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain selected from the sequences shown below, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the sequences shown below: SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17;

[0023] And / or, the anti-BCMA antibody or its antigen-binding fragment comprises a light chain selected from the sequences shown below, or a light chain having at least 80%, 85%, 90%, 95% or 99% identity with the sequences shown below: SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

[0024] In a further preferred embodiment of the present invention, wherein,

[0025] The anti-BCMA antibody or its antigen-binding fragment contains the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:12; or,

[0026] The anti-BCMA antibody or its antigen-binding fragment contains the heavy chain variable region shown in SEQ ID NO:10 and the light chain variable region shown in SEQ ID NO:13; or,

[0027] The anti-BCMA antibody or its antigen-binding fragment contains the heavy chain variable region shown in SEQ ID NO:11 and the light chain variable region shown in SEQ ID NO:14.

[0028] In a further preferred embodiment of the present invention, wherein,

[0029] The anti-BCMA antibody comprises the heavy chain shown in SEQ ID NO:15 and the light chain shown in SEQ ID NO:18; or,

[0030] The anti-BCMA antibody comprises the heavy chain shown in SEQ ID NO:16 and the light chain shown in SEQ ID NO:19; or,

[0031] The anti-BCMA antibody contains the heavy chain shown in SEQ ID NO:17 and the light chain shown in SEQ ID NO:20.

[0032] In a further preferred embodiment of the invention, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound is selected from antibody-drug conjugates of general formula (II) or their pharmaceutically acceptable salts or solvent compounds, or their tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof:

[0033]

[0034] Ab is selected from the above-mentioned anti-BCMA antibody or its antigen-binding fragment.

[0035] y is selected from 2-10, preferably 4-10, and even better 4, 6, 8 or 10.

[0036] In a further preferred embodiment of the invention, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound is selected from antibody-drug conjugates or their pharmaceutically acceptable salts or solvent compounds as shown in general formula (III):

[0037]

[0038] Ab is selected from the above-mentioned anti-BCMA antibody or its antigen-binding fragment.

[0039] y is selected from 2-10, preferably 4-10, and even better 4, 6, 8 or 10.

[0040] In a further preferred embodiment of the invention, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound is selected from the following structures:

[0041]

[0042]

[0043]

[0044] Wherein, y is selected from 2-10, preferably 4-10, and even more preferably 4, 6, 8 or 10.

[0045] The present invention also provides a method for preparing an antibody-drug conjugate as shown in general formula (I) or a pharmaceutically acceptable salt or solvent compound thereof, comprising the following steps:

[0046]

[0047] After reduction, Ab reacts with general formula (F) to give the compound shown in general formula (I);

[0048] Wherein, W is as defined above.

[0049] Ab is selected from the above-mentioned anti-BCMA antibody or its antigen-binding fragment; y is 1~20; preferably 4-10; more preferably 4, 6, 8 or 10.

[0050] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0051] On the other hand, the present invention provides a pharmaceutical use, which relates to the use of anti-BCMA antibody-drug conjugates or pharmaceutically acceptable salts or solvents of such antibody-drug conjugates or pharmaceutical compositions thereof in the preparation of medicaments for treating BCMA-mediated diseases or conditions.

[0052] In a preferred embodiment of the present invention, the BCMA-mediated disease or condition is selected from cancer or autoimmune diseases, wherein the cancer is preferably a BCMA-expressing cancer, more preferably lymphoma, leukemia or myeloma; the autoimmune disease is selected from lupus erythematosus, IgA nephropathy or rheumatoid arthritis.

[0053] The antibody-drug conjugates and their pharmaceutically acceptable salts or solvent compounds of the present invention have significant antitumor effects and good safety, as well as good in vivo metabolic activity and long duration of in vivo efficacy, showing broad prospects for clinical application. [Detailed Description of the Invention]

[0054] I. Terminology

[0055] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains.

[0056] The three-letter and single-letter codes for amino acids used in this application are as described in J. Biol. Chem, 243, p3558 (1968).

[0057] The term "antibody" as used in this application refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The composition and sequence of amino acids in the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0058] In this application, the variable region of the antibody light chain may further include a constant region of light chain, which contains human or mouse κ, λ chains or variants thereof.

[0059] In this application, the variable region of the antibody heavy chain may further include a constant region of heavy chain, which includes human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0060] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are known as the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment of this application conform to the known Kabat numbering rules and Kabat or ABM definition rules (http: / / bioinf.org.uk / abs / ).

[0061] The term "antigen-presenting cell" or "APC" refers to a cell that displays a foreign antigen complexed with MHC on its surface. T cells recognize this complex using T cell receptors (TCRs). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells.

[0062] The term "antigen presentation" refers to the process by which APCs capture antigens and make them available for recognition by T cells, for example, as a component of MHC-I / MHC-II conjugates.

[0063] The term "BCMA" includes any variant or isotype of BCMA naturally expressed by cells. The antibody of this application is cross-reactive with BCMA derived from non-human species. Alternatively, the antibody may be specific to human BCMA and may not exhibit cross-reactivity with other species. BCMA or any variant or isotype thereof may be isolated from cells or tissues that naturally express them, or generated by recombinant techniques using those techniques common in the art and described herein. Preferably, the anti-BCMA antibody targets human BCMA with a normal glycosylation pattern.

[0064] The term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods, the techniques and methods of which are well known in the art, such as:

[0065] 1. Antibodies isolated from transgenic, transchromosomal animals (e.g., mice) containing human immunoglobulin genes, or from fusion tumors prepared therefrom;

[0066] 2. Antibodies isolated from host cells transformed to express antibodies, such as transfected tumors;

[0067] 3. Antibodies isolated from recombinant human antibody libraries; and

[0068] 4. Antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences into other DNA sequences.

[0069] These recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations that occur during antibody maturation.

[0070] In this application, the term "mouse antibody" refers to a monoclonal antibody against human BCMA prepared in accordance with the knowledge and skills in the art. Preparation involves injecting the test subject with BCMA antigen, followed by isolating fusion tumors expressing antibodies with the desired sequence or functional characteristics. In a preferred embodiment of this application, the mouse BCMA antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

[0071] The term "human antibody" includes antibodies having variable and constant regions of human immunoglobulin sequences. Human antibodies in this application may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human backbone sequence (i.e., "humanized antibody").

[0072] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody. Humanized antibodies overcome the drawback of chimeric antibodies, which, due to carrying a large amount of mouse protein components, induce a strong immune response. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the variable region of the human antibody to maintain its activity.

[0073] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can mitigate the immune response induced by murine antibodies. To create a chimeric antibody, a fusion tumor secreting murine-specific monoclonal antibodies is selected. Then, the variable region gene is selected from mouse fusion tumor cells, followed by the selection of the constant region gene of a human antibody, as needed. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or their variants, preferably including the heavy chain constant region of human IgG1, IgG2, or IgG4, or using the IgG1 heavy chain constant region that enhances ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity through amino acid mutation.

[0074] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a mole basis, the antibody fragment retains at least 10% of the parent antibody's binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanoantibodies, domain antibodies, and multispecific antibodies. A review of engineered antibody variants is available in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.

[0075] A "Fab fragment" consists of a light chain, a heavy chain (CH1), and a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0076] The “Fc” region contains two heavy chain segments, including the CH2 and CH3 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.

[0077] The “Fab’ fragment” contains a light chain and a heavy chain portion containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby enabling the formation of interchain disulfide bonds between the two heavy chains of two Fab’ fragments to form the F(ab’)2 molecule.

[0078] The “F(ab')2 segment” contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

[0079] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0080] The term "multispecific antibody," used in its broadest sense, encompasses antibodies that exhibit multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments covalently or non-covalently linked, etc.

[0081] The term "single-chain antibody" refers to a single-chain recombinant protein composed of the heavy chain variable region (VH) and light chain variable region (VL) of an antibody linked by a linker peptide. It is the smallest antibody fragment with a complete antigen-binding site.

[0082] The term "domain antibody fragment" refers to an immunoglobulin fragment with immunological function containing only the heavy chain variable region or the light chain variable region. In some cases, two or more VH regions are covalently linked with peptide linkers to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same or different antigens.

[0083] The term "integration with BCMA" in this application refers to the ability to interact with human BCMA.

[0084] The term "antigen binding site" in this application refers to the three-dimensional spatial site recognized by the antibody or antigen binding fragment of this application.

[0085] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side by the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed by ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a distinctive spatial conformation. Methods for determining which epitopes are bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0086] As used herein, the terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, when human BCMA is used as the analyte and an antibody as the ligand, and the assay is performed in an instrument using surface plasma resonance (SPR) technology, the antibody binds to the predetermined antigen with an equilibrium dissociation constant (KD) of approximately less than 10⁻⁷ M or even smaller, and its affinity for the predetermined antigen is at least twice that for nonspecific antigens other than the predetermined antigen or closely related antigens (such as BSA). The term "antibody that recognizes an antigen" may be used interchangeably with the term "antibody that specifically binds."

[0087] The term "cross-reactivity" refers to the ability of the antibody of this application to bind to BCMA from different species. For example, the antibody of this application that binds to human BCMA can also bind to BCMA from another species. Cross-reactivity is measured by detecting the specific reactivity of the antigen with the purified antigen in binding assays (e.g., SPR and ELISA), or by binding to or functional interaction with physiologically BCMA-expressing cells. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasma resonance (SPR) analysis, or flow cytometry.

[0088] The terms “inhibition” and “blocking” are used interchangeably and encompass both partial and complete inhibition / blocking. Inhibition / blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in ligand binding affinity upon contact with an anti-BCMA antibody compared to ligands not contacted with an anti-BCMA antibody.

[0089] The term “growth inhibition” (e.g., involving cells) is intended to include any measurable reduction in cell growth.

[0090] The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to the stimulation of an immune response by a specific antigen (i.e., passive or adaptive). The term “inducing” in the context of inducing CDC or ADCC refers to stimulating a specific direct cell-killing mechanism.

[0091] The “ADCC” mentioned in this application, which stands for antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors by recognizing the Fc fragment of an antibody. The ADCC effector function of antibodies can be enhanced, reduced, or eliminated by modifying the Fc fragment of IgG. This modification refers to mutation in the constant region of the antibody's heavy chain.

[0092] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and readily available in the prior art, such as in Cold Spring Harbor's Guide to Antibody Experimentation, Chapters 5-8 and 15. For example, mice can be immunized with human BCMA or fragments thereof, and the resulting antibodies can be coated, purified, and sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to non-human CDR regions. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr, or from the Immunoglobulin Journal, 2001 ISBN012441351.

[0093] The engineered antibodies or antigen-binding fragments described in this application can be prepared and purified using conventional methods. The corresponding antibody cDNA sequence can be selected and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the FC region. Stable pure strains are obtained by expressing antibodies that specifically bind to human antigens. Positive pure strains are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting antibodies can be purified and collected using conventional techniques. The 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. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0094] The antibody referred to in this application is a monoclonal antibody. This monoclonal antibody refers to an antibody obtained from a single cell line, which is not limited to eukaryotic, prokaryotic, or phage cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained through recombination using techniques such as fusion tumor technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

[0095] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid contacts the cells. "Administration," "giving," and "treatment" also mean the in vitro and ex vivo treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or another cell. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0096] "Treatment" means administering an oral or topical therapeutic agent, such as an antibody containing any of the present application, to a patient who has symptoms of one or more diseases, and for whom the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also known as a "therapeutic effective dose") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this application (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease present in every patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0097] The term “consistently composed of” or variations thereof, used throughout the specification and the scope of the patent application, means that it includes all of the element or group of elements, and optionally includes other elements that are similar to or different in nature from the element, which do not significantly alter or introduce new properties of the specified administration regimen, method or composition.

[0098] The term "naturally occurring" as used in this application refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in organisms (including viruses) that can be isolated from natural sources and has not been intentionally modified artificially in a laboratory is considered naturally occurring.

[0099] An "effective dose" includes a dose sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a dose sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0100] "Exogenous" refers to substances that are produced outside of an organism, cell, or human body, depending on the context.

[0101] "Endogenous" refers to substances that are produced in cells, organisms, or the human body in accordance with their context.

[0102] Homology refers to the sequence similarity between two polynucleotide sequences or two polypeptides. Two compared sequences are homologous at positions occupied by the same bases or amino acid monomer subunits; for example, if every position in two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, sequences are compared when the highest percentage of homology is obtained.

[0103] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include their progeny. Therefore, the words “transformation” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened in the original transformed cells. “Optional” or “optionally” means that the event or environment described subsequently may, but does not have to, occur; this statement includes the possibility or absence of such an event or environment. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a specific sequence may, but does not have to, be present.

[0104] "Pharmaceutical composition" means containing one or more antibodies or antigen-binding fragments described herein, as well as other components such as physiological / pharmaceutical carriers and excipients. The purpose of pharmaceutical compositions is to facilitate drug delivery to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0105] "Pharmaceutical-grade salts" refer to the salts of the antibody-drug conjugates of this invention. These salts are safe and effective when used in mammals and possess the expected biological activity. The antibody-drug conjugates of this invention contain at least one amino group, thus allowing them to form salts with acids. Non-limiting examples of pharmaceutical-grade salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0106] "Solvent compound" refers to the antibody-drug conjugate compound of the present invention forming a pharmaceutically usable solvent compound with one or more solvent molecules. Non-limiting examples of solvent molecules include: water, ethanol, acetonitrile, isopropanol, and ethyl acetate.

[0107] When used in this invention, "cytotoxic drug" refers to a substance that inhibits cell function and / or causes cell death or damage.

[0108] "Microtubule inhibitors" are compounds that exert antitumor effects by interfering with cell mitosis by inhibiting the polymerization of tubulin or promoting its aggregation. Non-limiting examples include: maytansine derivatives, calichiomycin, taxanes, vincristine, colchicine, and salicylate / oligostatin / monomethyloligostatin E (MMAE) / monomethyloligostatin F (MMAF).

[0109] "Connector" refers to a chemical module containing a covalent bond or atomic chain to which an antibody is covalently attached to a drug. Non-limiting examples of connectors include: aryl, heteroaryl, PEG, polymethylene oxide, succinate, succinylamine, diglycolate, malonic acid ester, and hexylamine.

[0110] The "drug load" (DAR) is represented by y, which is the average number of cytotoxic drugs per antibody in general formula (A). In this invention, the drug load ranges from 1 to 20 cytotoxic drugs (D) per antibody. Antibody-drug conjugates of general formula (A) are collections of antibodies conjugated with a certain range (1-20) of cytotoxic drugs. The drug load (DAR) in antibody-drug conjugates from the conjugation reaction can be characterized by conventional methods, such as mass spectrometry, HPLC, and ELISA. These methods allow for the quantitative distribution of the antibody-drug conjugate on the y-value.

[0111] This invention also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) by referring to relevant literature. Commercially available deuterated starting materials can be used in the preparation of the deuterated forms of compounds of formula (I), or they can be synthesized using conventional techniques with deuterating reagents, including but not limited to deuterboranes, trideuterontetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0112] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of pharmaceutical compositions is to facilitate drug delivery to the body, enhance the absorption of the active ingredient, and thereby exert its biological activity. The preparation of conventional pharmaceutical compositions is described in the Chinese Pharmacopoeia.

[0113] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of the antibody-drug conjugate of the present invention, or a salt of the compound described in the present invention, which is safe and effective in mammalian use and has the intended biological activity. The ligand-drug conjugate of the present invention contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0114] The term "drug loading" refers to the average amount of cytotoxic drug loaded onto each antibody or its antigen fragment in a molecule of formula (V). It can also be expressed as the ratio of drug amount to antibody amount. The drug loading range can be 0-12 cytotoxic drugs (D) linked to each ligand, preferably 1-10. In embodiments of the present invention, the drug loading is expressed as y, also known as the DAR value, exemplarily the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average amount of drug per ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC characterization.

[0115] In some embodiments of the present invention, the cytotoxic drug is coupled to the N-terminal amino group and / or the ε-amino group of the lysine residue of the ligand via a linker unit. In other embodiments of the present invention, the cytotoxic drug is coupled to the thiol group of the ligand via a linker unit. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.

[0116] The loading of ligand-cytotoxic drug conjugates can be controlled using the following non-limiting methods, including:

[0117] (1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

[0118] (2) Control the reaction time and temperature.

[0119] (3) Choose different reaction reagents.

[0120] The antibody-drug conjugates of the present invention, or their pharmaceutically acceptable salts or solvent compounds, have significant antitumor effects and good safety profiles. Implementation

[0121] The following embodiments are used to further describe this application, but these embodiments are not intended to limit the scope of this application. Experimental methods not specified in the embodiments of this application are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Selection Manual; or under conditions recommended by the raw material or product manufacturer.

[0122] Experimental methods in this invention, where specific conditions are not specified, are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0123] The known raw materials of this invention can be synthesized using or according to methods known in the art, or can be purchased from companies such as Jiangsu Aikon, as detailed in the table below:

[0124]

[0125] [Example 1-1 Synthesis of Compound a]

[0126]

[0127] Place raw material a-1 (4.1 g, 9.71 mmol) and raw material a-2 (4.3 g, 8.09 mmol, containing 4% amino isomer impurities) in a 250 mL reaction flask. Under nitrogen protection, add DCM (54 mL) and MeOH (18 mL), stir and cool to 0 °C. Add DMTMM (3.6 g, 12.1 mmol) and triethylamine (2.5 g, 24.2 mmol), and maintain the reaction at 0 °C with stirring for 1 h. HPLC monitoring shows that the reaction of raw material a-2 is complete. Evaporate the reaction solution to dryness under reduced pressure (<25 °C), add MTBE (120 mL), stir and slurry (to form a slurry), pour off the solution and filter. Add another 120 mL of the slurry. MTBE was pulped (solid), filtered, and the filter cake was washed with water (60 mL × 2). The crude product was dried and dissolved in dichloromethane and methanol. The solution was then subjected to two wet column chromatography analyses (using DCM:MeOH extractant = 40:1-20:1) to obtain compound a (6.2 g, 7.37 mmol), purity: 99.3%, yield: 91%.

[0128] [Synthesis of Compound b in Examples 1-2]

[0129]

[0130] Compound a (5.7 g, 6.77 mmol) was placed in a 500 mL three-necked flask. Under nitrogen protection, dry THF (114 mL) was added, stirred to dissolve, and the internal temperature was cooled to about -10 °C. DBU (3.09 g, 20.31 mmol) was added, and the internal temperature was maintained at -10 to -5 °C during the dropwise addition. The addition was completed in 5 min. After the dropwise addition was completed, the internal temperature was maintained at -10 to -5 °C, and the reaction was allowed to proceed for 2.5 h. The solid precipitated out.

[0131] Cool the internal temperature to -20℃, add MTBE (114mL), and maintain the internal temperature between -20℃ and -10℃ during the process. The product is completely precipitated. Filter, wash the filter cake with MTBE (57mL×2), and dry it to obtain 6g of crude compound b. Store at -78℃ for later use.

[0132] [Synthesis of Compound e in Examples 1-3]

[0133]

[0134] Compound c (651 mg, 1 mmol) was dissolved in 10 mL of DCM. Stirring was started while the mixture was cooled in an ice bath, and DBU (456 mg, 3 mmol) was added dropwise. After reacting for one hour in an ice bath, the reaction was complete. Compound d (257 mg, 1 mmol) and HATU (420 mg, 1.1 mmol) were added sequentially. After stirring in an ice bath for 30 minutes, LC-MS showed that the reaction was complete. The reaction solution was concentrated at 25 °C, and the residue was purified by reverse-phase column chromatography (ACN in H2O, 50% product yield) to give compound e, a reddish-brown solid, 130 mg, yield 19%.

[0135] MS:691.3[M+23].

[0136] [Synthesis of Compound f in Examples 1-4]

[0137]

[0138] Compound e (130 mg, 0.19 mmol) was dissolved in DCM, and anisole (62 mg, 0.57 mmol) and dichloroacetic acid (245 mg, 1.9 mmol) were added. The reaction was stirred overnight at room temperature for a total of 16 hours. The LC-MS sample was taken and showed that the starting material was completely consumed. The reaction was stopped, and the reaction solution was concentrated at 25 °C. The residue was purified by reverse-phase column chromatography (ACN / H2O, 30% product yield) to give compound f, a pink solid, 53 mg, with a yield of 54%.

[0139] MS:519.2[M+1].

[0140] [Examples 1-5: Synthesis of Compound D]

[0141]

[0142] Compound f (23 mg, 0.044 mmol) and compound b (27 mg, 0.044 mmol) were dissolved in DCM (3 mL) and MeOH (1 mL) and cooled to -30 °C under nitrogen protection. DMTMM (20 mg, 0.067 mmol) was added, and the reaction was carried out at -20 °C to -10 °C for 1 hour. LC-MS analysis showed complete consumption of the starting materials. The reaction was quenched with 10 mL of water at -10 °C, followed by the addition of 30 mL of DCM for layering. The aqueous phase was extracted with a DCM / MeOH ratio of 10 / 1 (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 25 °C. The residue was purified using a preparative method (ACN / H₂O / 0.05% FA) to give compound D, a white solid, 5.8 mg, yield 12%, HPLC purity 98.87%.

[0143] MS:1120.3[M+1].

[0144] [Example 2: Antigen Preparation]

[0145] The human BCMA (BCMA-His) protein encoding the His tag was synthesized by SinoBiologics (Cat No.:Cat No.:10620-H08H).

[0146] BCMA-His sequence:

[0147] MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAHHHHHHHHHHHH SEQ ID NO:21

[0148] [Example 3: Obtaining the mouse fusion tumor and antibody sequence]

[0149] Animals were immunized with human antigen BCMA-His. Five Balb / c mice and five A / J mice, female and 10 weeks old, were used. The immunogen and immune adjuvant were fully mixed and emulsified in a 1:1 ratio to prepare a stable "water-in-oil" liquid. The injection dose was 25 μg / 200 μL / mouse.

[0150] Table 1. Immunization regimen

[0151] Serum from immunized mice was assessed for serum titer and binding capacity to cell surface antigens using the indirect ELISA method described in Example 3. Cell fusion was initiated based on control titer (greater than 100,000-fold dilution). Mice with high serum titer, affinity, and FACS binding were selected for a final immunization, followed by sacrifice. Spleen cells and SP2 / 0 myeloma cells were fused and plated to obtain fusion tumors. Target fusion tumors were screened using indirect ELISA, and single cell lines were established using limiting dilution. Positive antibody lines were further screened using indirect ELISA to select fusion tumors binding recombinant proteins. Log-phase fusion tumor cells were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript™ Reverse Transcriptase, Takara #2680A). The cDNA obtained from reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and sequenced to obtain the sequence of the mouse antibody M1.

[0152] The heavy and light chain variable region sequences of mouse monoclonal antibody M1 are as follows:

[0153] M1 HCVR

[0154] SEQ ID NO:1

[0155] M1 LCVR

[0156] EILLTQSPAIIVTSPGEKVTITCSASSSVIYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFSFTINSMEAEDVATYYCQQRSSYPLTFGAGTKLELK SEQ ID NO:2

[0157] Table 2. CDR sequences of heavy and light chain variable regions of mouse monoclonal antibody M1

[0158] [Example 4: Method for detecting the in vitro binding activity of antibodies]

[0159] (1) In vitro indirect ELISA binding assay:

[0160] Dilute BCMA His protein (Sino Biological Inc., cat# 10428-H08H) to a concentration of 1 μg / ml with PBS at pH 7.4, and add 100 μl / well to each well of a 96-well high-affinity microplate. Incubate overnight (16-20 hours) at 4°C. Wash the plate four times with PBST (pH 7.4 PBS containing 0.05% Tween-20), then add 150 μl / well of 3% bovine serum albumin (BSA) blocking buffer diluted with PBST and incubate at room temperature for 1 hour to block. After blocking, discard the blocking buffer and wash the plate four times with PBST buffer.

[0161] Dilute the antibody to be tested with PBST containing 3% BSA, starting at 1 μM, in a 10-fold gradient, 10 doses, and add 100 μl / well to the microplate. Incubate at room temperature for 1 hour. After incubation, wash the plate 4 times with PBST, add 100 μl / well of HRP-labeled goat anti-human secondary antibody (Abeam, cat#ab97225) diluted with PBST containing 3% BSA, and incubate at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100 μl / well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature in the dark for 1 minute, and stop the reaction with 100 μl / well of Stop Solution (Cell Signaling Technology, cat#7002S). Read the absorbance at 450 nm using a microplate reader (BioTek, model Synergy H1) and analyze the data. The results of concentration signal curve analysis are shown in the table below:

[0162] Table 3. Affinity (EC50 value) of mouse antibodies to human BCMA antigen

[0163] (2) In vitro cell binding experiment:

[0164] Collect cultured BCMA-overexpressing cells (HEK-293T cells overexpressing BCMA and BCMA-expressing tumor cells, NCI-H929), adjust cell density, and seed them into 96-well U-plates at 1×10⁵ to 2×10⁵ cells per well. Centrifuge at 1200g for 5 min, discard the supernatant, add 100 μL of serially diluted antibody solution or mouse immune serum, and incubate at 4°C for 60 min. Centrifuge at 1200g for 5 min, discard the supernatant, wash cells twice with PBS, add 100 μL of fluorescently labeled secondary antibody (PE-labeled goat anti-human monoclonal antibody or PE-labeled goat anti-mouse monoclonal antibody) per well, and incubate at 4°C for 60 min. Centrifuge at 1200g for 5 min, discard the supernatant. Wash cells twice with PBS, resuspend in PBS, detect signal using flow cytometry, and analyze the concentration curve results.

[0165] Table 4. Affinity (EC50 value) of mouse antibodies to cells expressing BCMA

[0166] [Example 5: Mouse Antibody Humanization Experiment]

[0167] The humanization of mouse anti-human BCMA monoclonal antibodies was performed using methods disclosed in many publications in the field. In short, by replacing the parental (mouse antibody) constant domain with a human constant domain and selecting a human antibody sequence based on the homology between the mouse antibody and the human antibody, this application humanized mouse antibody M1.

[0168] Based on the typical structure of the mouse antibody VH / VL CDR obtained, the variable region sequences of the heavy and light chains were compared with the human antibody germline database to obtain a human germline template with high homology.

[0169] The CDR region of the murine antibody M1 was transplanted into a selected humanized template. Then, based on the three-dimensional structure of the murine antibody, reverse mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH. Chemically unstable amino acid residues in the CDR region were optimized. After expression testing and comparison of the number of reverse mutations, the sequence for designing the humanized heavy chain variable region HCVR was selected. The sequence is as follows:

[0170] HCVR1

[0171] SEQ ID NO:9

[0172] HCVR2

[0173] SEQ ID NO:10

[0174] HCVR3

[0175] SEQ ID NO:11

[0176] The sequences with humanized light chain variable region LCVR were selected, and the sequences are as follows:

[0177] LCVR1

[0178] EIVLTQSPATLSLSPGERATLSCSASSSVIYMNWYQQKPGQAPRLLIYGISNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK SEQ ID NO:12

[0179] LCVR2

[0180] EIVLTQSPATLSLSPGERATLSCSASSSVIYMNWYQQKPGQSPKIWIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK SEQ ID NO:13

[0181] LCVR3

[0182] EILLTQSPATLSLSPGERATLTCSASSSVIYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFTLTISSLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK SEQ ID NO:14

[0183] The designed heavy and light chain variable region sequences were linked to the human IgG1 heavy chain and human antibody light chain constant region sequences, as exemplarily shown below:

[0184] IgG1 C

[0185] SEQ ID NO:22

[0186] Ig kappa C

[0187] SEQ ID NO:23

[0188] The heavy chain and light chain sequences are as follows:

[0189] Ab1 HC

[0190] SEQ ID NO:15

[0191] Ab2 HC

[0192] SEQ ID NO:16

[0193] Ab3 HC

[0194] SEQ ID NO:17

[0195] Ab1 LC

[0196] SEQ ID NO:18

[0197] Ab2 LC

[0198] SEQ ID NO:19

[0199] Ab3 LC

[0200] SEQ ID NO:20

[0201] Table 5. Sequence numbers of antibodies and their heavy chain, light chain, and variable regions

[0202] Based on the amino acid sequences of the light and heavy chains of the above-mentioned humanized antibodies, cDNA fragments were synthesized and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). HEK293 cells (Life Technologies Cat. No. 11625019) were transfected with the expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) at a 1:2 ratio and incubated in a CO2 incubator for 4-5 days. The cell culture medium was collected, centrifuged, filtered, and loaded onto an antibody purification affinity column. The column was washed with phosphate buffer, extracted with glycine-buffered saline (pH 2.7 0.1M Gly-HCl), neutralized with 1M Tris hydrochloric acid at pH 9.0, and dialyzed against phosphate buffer to obtain the humanized antibody protein of this application.

[0203] [Example 6: In vitro binding affinity and kinetics experiment]

[0204] The affinity (EC50) of each humanized antibody for human BCMA antigen, as determined by in vitro indirect ELISA using Example 4(1), is shown in the table below:

[0205] Table 6. Affinity (EC50) of each humanized antibody to human BCMA antigen

[0206] The affinity (EC50) of each humanized antibody for NCI-H929 tumor cells, as determined by the in vitro cell binding assay in Example 4(2), is shown in the table below:

[0207] Table 7. Affinity (EC50) of each humanized antibody to NCI-H929 tumor cells

[0208] [Example 7: Antibody endocytosis]

[0209] To determine whether the antibody of this invention, after binding to BCMA, can be co-endocytosed into cells along with human BCMA, NCI-H929 cells were used for evaluation. NCI-H929 cells were digested with trypsin (first washed with PBS, 37°C, approximately 2 min), and the cells were collected and resuspended in pre-chilled FACS buffer, adjusting the cell concentration to 1×10⁶ / mL. In an EP tube, 1 mL of cell suspension was added, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. 1 mL of the prepared antibody was added to resuspend the cells, with a final antibody concentration of 20 μg / mL. The cells were incubated on a shaker at 4°C for 1 h, centrifuged and the supernatant discarded (4°C, 1500 rpm × 5 min), washed twice with FACS buffer, and the supernatant discarded. 100 μL of fluorescent secondary antibody working solution was added to each tube to resuspend the cells, incubated on a shaker at 4°C for 30 min, centrifuged and the supernatant discarded (4°C, 1500 rpm × 5 min), washed twice with FACS buffer, and the supernatant discarded. Add 1.0 mL of preheated NCI-H929 complete cell culture medium to each tube to resuspend the cells and mix well. Aliquot into 4 tubes, 200 μL each, for the 0 min, blank, 30 min, and 2 h groups, respectively. Remove the 0 min and blank tubes and place them on ice. Incubate the remaining tubes at 37°C and perform endocytosis for 30 min and 2 h, respectively. At the corresponding time points, remove the EP tubes and pre-cool them on ice for 5 min. Centrifuge all treatment groups (4°C, 1500 rpm × 5 min) and discard the supernatant. Wash once with FACS buffer and discard the supernatant. Add 250 μL of strip buffer to the EP tubes of all treatment groups except the 0 min group, incubate at room temperature for 8 min, centrifuge and discard the supernatant (4°C, 1500 rpm × 5 min), wash twice with FACS buffer and discard the supernatant. Add 100 μL of immunostaining fixative to all treatment groups, incubate at 4°C for at least 30 min, and analyze using a DxFlex flow cytometer. BCMA antibody endocytosis percentage = (fluorescence intensity values ​​at each time point - average fluorescence intensity value at zero point) / average fluorescence intensity value at zero point. Results are shown in the table below:

[0210] Table 8. Internalization of antibodies in NCI-H929 tumor cells (EC50)

[0211] [Example 8: Preparation of antibody-drug conjugates]

[0212] Preparation of ADC2

[0213]

[0214] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10mM, 0.347mL, 3.47μmol) was added to the PBS buffered aqueous solution of antibody Ab2 (pH=6.5, 0.05M PBS buffered aqueous solution; 7.3ml, 13.8mg / ml, 0.681μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 14.0ml, and 3.3ml of the solution was taken out and used for further reaction.

[0215] compound [D] (3.0 mg, 3.72 μmol) was dissolved in 0.15 mL of DMSO and added to the above 3.3 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (extraction phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product of the Ab2 antibody conjugate, ADC2, in PBS buffer (1.35 mg / mL, 13 mL), which was then frozen at 4 °C.

[0216] The average value y was determined using the ultraviolet method. After placing cuvettes containing sodium succinate buffer into the reference absorption cell and the sample absorption cell, respectively, and subtracting the solvent blank, the cuvettes containing the test solution were placed into the sample absorption cell, and the absorbance at 280 nm and 370 nm was measured.

[0217] Data processing:

[0218] By establishing a standard curve and measuring the absorption at a wavelength of 280 nm, the antibody content CAb was determined, and the small molecule content CDrug was determined by measuring the absorption at a wavelength of 370 nm.

[0219] The average drug load is y = CDrug / CAb.

[0220] The y-value of the exemplary product ADC2 was determined to be 4 using the above method. The ADC2 (y=4) sample was obtained by UV-HPLC purification.

[0221] [Example 9: Cell-killing activity of antibody-drug conjugates]

[0222] To detect the cytotoxic effect of the antibody-drug conjugate disclosed herein on tumor cells, the BCMA-high expression level cell line NCI-H929 (ATCC accession number CRL-9068) was used for evaluation. NCI-H929 cells were collected, centrifuged, counted, and then adjusted to a cell density of 1×10⁵ cells / mL with complete culture medium. 100 μL of the medium was seeded into the center 60 wells of a white 96-well plate, resulting in 10,000 cells / well. 100 μL of DPBS was added to the edge wells. The cell culture plates were incubated overnight at 37°C in a 5% CO₂ incubator. The next day, the antibody-drug conjugate working solution was prepared in a 96-well V-type plate using complete culture medium, starting at 133 μM, diluted 5-fold, resulting in 9 concentrations. 80 μL of the solution was added to each well of the white 96-well plate, with two replicates. The cell culture plates were then incubated at 37°C in a 5% CO₂ incubator for 72 hours. On day 5 of the experiment, the cell culture plates were removed and brought to room temperature. 90 μl of CellTiter-Glo® cell viability assay reagent (Promega, Cat#: G7573) was added to each well. After vortexing and mixing, the plates were incubated in the dark for 10 minutes before being analyzed using the chemiluminescence immunoassay program on a microplate reader. The IC50 value was calculated using GraphPad Prims software. The negative antibody IgG1 and the compound were used... [D] The antibody-drug conjugate (y=4) was used as a negative control. The experimental results are shown in the table below:

[0223] Table 9. Killing effect of antibody-drug conjugates on tumor cells

[0224] Experimental results show that ADC2 (y=4) has good in vitro killing activity against tumor cells.

[0225] [Example 10: Tumor-killing activity of antibody-drug conjugates]

[0226] To further investigate the inhibitory effect of the antibody-drug conjugate on the proliferation of tumors formed in vivo, the antitumor effect of the antibody-drug conjugate disclosed herein was evaluated after xenografts were formed in mice using NCI-H929 cells (ATCC accession number CRL-9068). 10 x 10⁶ NCI-929 cells were subcutaneously inoculated into the back of 6-8 week old immunodeficient female mice (NOD SCID). The antibody-drug conjugate was administered intravenously 3 mg / kg once weekly via tail vein injection for two weeks, starting 14 days later. The control group received human IgG1 isotype control protein at a dose of 3 mg / kg. Each control and treatment group consisted of 5 mice. The tumor inhibition rate was calculated by measuring tumor volume using the following formula:

[0227] Tumor inhibition rate (TGI) % = 100% - (tumor volume of the treatment group on day 14 - tumor volume of the treatment group on day 0) / (tumor volume of the control group on day 14 - tumor volume of the control group on day 0).

[0228] The experimental results are shown in the table below:

[0229] Table 10. Antibody-drug conjugates' tumor-killing effects

[0230] Experimental results show that the antibody-drug conjugate ADC2 (y=4) exhibits good in vivo tumor inhibition effects.

[0231] <110> Shanghai Hansoh Biomedical Co., Ltd. (a mainland Chinese company) and Jiangsu Hansoh Pharmaceutical Group Co., Ltd. (a mainland Chinese company)

[0232] <120> An antibody-drug conjugate and its pharmaceutical uses

[0233] <130>

[0234] <150> CN 202110123809.3

[0235] <151> 2021-01-29

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[0343] <221> domain

[0344] <222> (1)..(121)

[0345] <223> Humanized antibody heavy chain variable region 3

[0346] <400> 11

[0347]

[0348] <210> 12

[0349] <211> 106

[0350] <212> PRT

[0351] <213> Artificial Sequence

[0352] <220>

[0353] <221> domain

[0354] <222> (1)..(106)

[0355] <223> Humanized antibody light chain variable region 1

[0356] <400> 12

[0357]

[0358] <210> 13

[0359] <211> 106

[0360] <212> PRT

[0361] <213> Artificial Sequence

[0362] <220>

[0363] <221> domain

[0364] <222> (1)..(106)

[0365] <223> Humanized antibody light chain variable region 2

[0366] <400> 13

[0367]

[0368] <210> 14

[0369] <211> 106

[0370] <212> PRT

[0371] <213> Artificial Sequence

[0372] <220>

[0373] <221> domain

[0374] <222> (1)..(106)

[0375] <223> Humanized antibody light chain variable region 3

[0376] <400> 14

[0377]

[0378] <210> 15

[0379] <211> 451

[0380] <212> PRT

[0381] <213> Artificial Sequence

[0382] <220>

[0383] <221> chain

[0384] <222> (1)..(451)

[0385] <223> Humanized antibody heavy chain 1

[0386] <400> 15

[0387]

[0388]

[0389] <210> 16

[0390] <211> 451

[0391] <212> PRT

[0392] <213> Artificial Sequence

[0393] <220>

[0394] <221> chain

[0395] <222> (1)..(451)

[0396] <223> Humanized antibody heavy chain 2

[0397] <400> 16

[0398]

[0399]

[0400] <210> 17

[0401] <211> 451

[0402] <212> PRT

[0403] <213> Artificial Sequence

[0404] <220>

[0405] <221> chain

[0406] <222> (1)..(451)

[0407] <223> Humanized antibody heavy chain 3

[0408] <400> 17

[0409]

[0410]

[0411] <210> 18

[0412] <211> 213

[0413] <212> PRT

[0414] <213> Artificial Sequence

[0415] <220>

[0416] <221> chain

[0417] <222> (1)..(213)

[0418] <223> Humanized antibody light chain 1

[0419] <400> 18

[0420]

[0421]

[0422] <210> 19

[0423] <211> 213

[0424] <212> PRT

[0425] <213> Artificial Sequence

[0426] <220>

[0427] <221> chain

[0428] <222> (1)..(213)

[0429] <223> Humanized antibody light chain 2

[0430] <400> 19

[0431]

[0432] <210> 20

[0433] <211> 213

[0434] <212> PRT

[0435] <213> Artificial Sequence

[0436] <220>

[0437] <221> chain

[0438] <222> (1)..(213)

[0439] <223> Humanized antibody light chain 3

[0440] <400> 20

[0441]

[0442] <210> twenty one

[0443] <211> 65

[0444] <212> PRT

[0445] <213> Artificial Sequence

[0446] <220>

[0447] <221> peptides

[0448] <222> (1)..(65)

[0449] <223> Human BCMA-His protein

[0450] <400> twenty one

[0451]

[0452]

[0453] <210> twenty two

[0454] <211> 330

[0455] <212> PRT

[0456] <213> Artificial Sequence

[0457] <220>

[0458] <221> domain

[0459] <222> (1)..(330)

[0460] <223> Heavy chain constant region

[0461] <400> twenty two

[0462]

[0463]

[0464] <210> twenty three

[0465] <211> 107

[0466] <212> PRT

[0467] <213> Artificial Sequence

[0468] <220>

[0469] <221> domain

[0470] <222> (1)..(107)

[0471] <223> Light chain constant region

[0472] <400> twenty three

[0473]

Claims

1. A compound represented by general formula (F), wherein, W is selected from -(CReRf)g-X1-(CReRf)u-X2-(CReRf)h-, Re or Rf is independently selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl or hydroxyalkyl, X1 or X2 is independently selected from O or S, and g, u or h is independently selected from 1, 2, 3 or 4.

2. The compound as claimed in claim 1, wherein, Re or Rf are each independently selected from hydrogen and deuterium, X1 or X2 are each independently selected from S and O, and g, u or h are each independently selected from 1, 2, and 3.

3. The compound as claimed in claim 2, wherein the compound is selected from:

4. A method for preparing an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof, comprising conjugating an antibody with a compound as claimed in claims 1 to 3.

5. An antibody-drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, Ab is an antibody or its antigen-binding fragment, W is selected from -(CReRf)g-X1-(CReRf)u-X2-(CReRf)h-, Re or Rf is independently selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl or hydroxyalkyl, X1 or X2 is independently selected from O or S, g, u or h is independently selected from 1, 2, 3 or 4, and y is 1 to 20.

6. The antibody-drug conjugate as claimed in claim 5, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, Re or Rf are each independently selected from hydrogen or deuterium, X1 or X2 are each independently selected from S or O, g, u or h are each independently selected from 1, 2, or 3, and y is 4 to 10.

7. The antibody-drug conjugate as claimed in claim 6, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, Re or Rf represents hydrogen, X1 or X2 represents O, g, u or h represents 2, and y represents 4, 6, 8 or 10.

8. The antibody-drug conjugate as described in any one of claims 5 to 7, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, Ab is an anti-BCMA antibody or its antigen-binding fragment, which includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO:

8.

9. The antibody-drug conjugate as claimed in claim 8, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment is selected from murine antibodies, chimeric antibodies, human antibodies, or humanized antibodies.

10. The antibody-drug conjugate as claimed in claim 9, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

11. The antibody-drug conjugate as claimed in claim 10, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment contains the heavy chain constant region of human IgG1, IgG2, or IgG4.

12. The antibody-drug conjugate as claimed in claim 11, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment contains a constant region of the IgG1 heavy chain that exhibits enhanced ADCC toxicity after amino acid mutation.

13. The antibody-drug conjugate as claimed in claim 12, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment contains a heavy chain constant region as shown in SEQ ID NO:

22.

14. The antibody-drug conjugate as claimed in claim 9, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment further comprises a light chain constant region of the human antibody κ chain, λ chain, or a variant thereof.

15. The antibody-drug conjugate as claimed in claim 14, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment contains the light chain constant region of the human antibody κ chain.

16. The antibody-drug conjugate as claimed in claim 15, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment contains a light chain constant region as shown in SEQ ID NO:

23.

17. The antibody-drug conjugate as claimed in claim 9, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the sequences shown below: SEQ ID NO: 10, SEQ ID NO: 9, or SEQ ID NO: 11; and / or a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the sequences shown below: SEQ ID NO: 13, SEQ ID NO: 12, or SEQ ID NO:

14.

18. The antibody-drug conjugate as claimed in claim 17, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment contains the heavy chain variable region shown in SEQ ID NO: 10 and the light chain variable region shown in SEQ ID NO: 13; or, the anti-BCMA antibody or its antigen-binding fragment contains the heavy chain variable region shown in SEQ ID NO: 9 and the light chain variable region shown in SEQ ID NO: 12; or, the anti-BCMA antibody or its antigen-binding fragment contains the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO:

14.

19. The antibody-drug conjugate as claimed in claim 17, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain selected from the sequences shown below, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the sequences shown below: SEQ ID NO: 16, SEQ ID NO: 15, or SEQ ID NO: 17; and / or a light chain selected from the sequences shown below, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with the sequences shown below: SEQ ID NO: 19, SEQ ID NO: 18, or SEQ ID NO:

20.

20. The antibody-drug conjugate as claimed in claim 19, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The anti-BCMA antibody comprises the heavy chain shown in SEQ ID NO: 16 and the light chain shown in SEQ ID NO: 19; or, the anti-BCMA antibody comprises the heavy chain shown in SEQ ID NO: 15 and the light chain shown in SEQ ID NO: 18; or, the anti-BCMA antibody comprises the heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO:

20.

21. The antibody-drug conjugate as claimed in claim 5, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, selected from the antibody-drug conjugate as shown in general formula (II), or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof: wherein, Ab, y are as defined in any of the requests 5 to 7.

22. The antibody-drug conjugate as claimed in claim 21, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, selected from the antibody-drug conjugate of general formula (III), or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof: wherein, Ab, y are as defined in any of the requests 5 to 7.

23. The antibody-drug conjugate as described in any one of claims 5 to 22, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, wherein, The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound is selected from the following structures: wherein Ab1, Ab2, and Ab3 are anti-BCMA antibodies or antigen-binding fragments, the anti-BCMA antibody or antigen-binding fragments comprising a heavy chain variable region and a light chain variable region, wherein Ab1 comprises a heavy chain variable region as shown in SEQ ID NO: 9 and a light chain variable region as shown in SEQ ID NO: 12; Ab2 comprises a heavy chain variable region as shown in SEQ ID NO: 10 and a light chain variable region as shown in SEQ ID NO: 13; Ab3 comprises a heavy chain variable region as shown in SEQ ID NO: 11 and a light chain variable region as shown in SEQ ID NO: 13; and y is as defined in any one of claims 5 to 7.

24. A method for preparing an antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt or solvent compound thereof, comprising the steps of: After reduction, Ab is coupled with general formula (F) to give a compound of general formula (I), wherein W, Ab, and y are as defined in any one of claims 5 to 7.

25. A pharmaceutical composition comprising, as described in any one of claims 5 to 23, an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

26. Use of any antibody-drug conjugate as claimed in any one of claims 5 to 23, or a pharmaceutically acceptable salt or solvent compound thereof, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutical composition as claimed in claim 25, in the preparation of a medicament for treating BCMA-mediated diseases or conditions.

27. The use as described in claim 26, wherein, The BCMA-mediated disease or condition is selected from cancer or autoimmune diseases.

28. The use as described in claim 27, wherein, This cancer expresses BCMA.

29. The use as described in claim 28, wherein, Cancers that express BCMA include lymphoma, leukemia, or myeloma.

30. The use as described in claim 27, wherein, The autoimmune disease is lupus, IgA nephropathy, or rheumatoid arthritis.

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