Antibody-drug conjugate and use thereof

Abstract

Provided is an antibody-drug conjugate having a structure represented by formula (I): Ab-(L-D)n (I), wherein Ab is an anti-interleukin 6 receptor antibody or an antigen-binding fragment thereof, D is a drug, L represents a linking group for linking Ab and D, and n is 1-10. The antibody or the antigen-binding fragment thereof comprises at least one light chain and at least one heavy chain. In addition, provided is use of the antibody-drug conjugate. The antibody-drug conjugate can significantly reduce the administration dosage, improve the tolerance of patients, and reduce adverse reactions.

Description

Antibody-drug conjugates and their uses Technical Field

[0001] This application relates to the field of antibody-drug conjugates, and more particularly to an antibody-drug conjugate formed by conjugating an antibody or antigen-binding fragment against an interleukin-6 receptor with a drug, as well as its preparation and use. Background Technology

[0002] Antibody-drug conjugates (ADCs) are created by linking an antibody or antibody fragment (targeting portion) to a small molecule drug (payload), such as a glucocorticoid, via a linker. This leverages the cell-specific targeting of the antibody and the activity of the small molecule drug. Currently, the vast majority of ADCs are formed by linking antibodies targeting tumor antigens to small molecule chemical drugs via linkers. By utilizing the specific binding characteristic of antibodies to target antigens, the small molecule drug is delivered directly to the desired site.

[0003] Human interleukin-6 (IL-6) is a 26 kDa glycoprotein whose encoding gene is located on chromosome 7. The functional diversity of IL-6 mainly lies in its interaction with various IL-6 receptors on the cell surface, and then the transmission of various biological signals to different tissues and cells via downstream signal transduction pathways.

[0004] Interleukin-6 receptor (IL-6R) plays a crucial role in immune regulation, hematopoiesis, inflammation, and tumorigenesis. Studies have shown that IL-6R is highly expressed in various human tumor cells, including those of promyelocytic leukemia, astrocytoma, glioblastoma, multiple myeloma, prostate cancer, gastric cancer, and liver cancer cells. Therefore, IL-6 pathway inhibitors, which target IL-6 or IL-6R to block the binding of IL-6 to IL-6R and thus inhibit IL-6 pathway signaling, have become potential therapeutic approaches for various diseases.

[0005] Glucocorticoid receptor agonists are a class of potent small molecules used to treat inflammatory diseases, but their efficacy in the chronic treatment of these diseases is limited due to serious side effects, and larger effective doses also cause poor tolerability in patients. Therefore, there is a need to develop drugs with minimal adverse effects and improved patient tolerability. Summary of the Invention

[0006] This application relates to an antibody-drug conjugate, its preparation method, its use, and a pharmaceutical composition comprising the antibody-drug conjugate.

[0007] In a first aspect, the present invention provides an antibody-drug conjugate having a structure represented by formula (I): Ab-(LD)n (I)

[0008] in:

[0009] Ab is an antibody against interleukin-6 receptor or its antigen-binding fragment, said antibody or its antigen-binding fragment comprising at least one light chain and at least one heavy chain, said light chain comprising at least one light chain variable region, said heavy chain comprising at least one heavy chain variable region; said heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 HCDR1, an amino acid sequence as shown in general formula (1) HCDR2, and an amino acid sequence as shown in SEQ ID NO:3 HCDR3; said light chain variable region having an amino acid sequence as shown in SEQ ID NO:4 LCDR1, an amino acid sequence as shown in SEQ ID NO:5 LCDR2, and an amino acid sequence as shown in SEQ ID NO:6 LCDR3; said general formula (1) has an amino acid sequence as shown in FISYSGX1TTYNPSLKS, wherein X1 is selected from G, V, F, K or R.

[0010] D represents a drug.

[0011] L represents the linking group used to connect Ab and D, and

[0012] n is 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0013] In some embodiments, the HCDR2 of the heavy chain variable region has an amino acid sequence as described in any one of SEQ ID NO:7, 9, 16, 19 and 20.

[0014] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 25, 27, 34, 37 or 38, or an amino acid sequence having at least 85% sequence identity with it.

[0015] In some embodiments, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:60 or an amino acid sequence having at least 85% sequence identity with it.

[0016] In some embodiments, the Fc region of the antibody may have one or more mutations to prolong the antibody's half-life. In some embodiments, the Fc region of the antibody may have mutations at one or more of positions 252, 254, and 256. In some embodiments, the Fc region of the antibody has one or more mutations of M252Y, S254T, and T256E.

[0017] In some embodiments, the heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO:62-66 or an amino acid sequence having at least 85% sequence identity with it.

[0018] In some embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:61 or an amino acid sequence having at least 85% sequence identity with it.

[0019] In some embodiments, the antibody is a humanized antibody.

[0020] In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody.

[0021] In some embodiments, the antibody is an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody.

[0022] In some embodiments, the antibody or antigen-binding fragment has good thermal stability and freeze-thaw stability.

[0023] In some embodiments, the isoelectric point range of the antibody or antigen-binding fragment may be 9.0 to 10.0.

[0024] In some embodiments, the drug (D) linked to the antibody is a glucocorticoid.

[0025] In some embodiments, the drug (D) linked to the antibody may be a modified glucocorticoid.

[0026] In some embodiments, the drug (D) linked to the antibody is hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, and modified forms of these drugs.

[0027] In some embodiments, the drug (D) linked to the antibody may have the following formula (D1) or (D2):

[0028] R1 can be H, deuterium, or -H2PO3, and each time R appears, it is independently H, deuterium, or a halogen (e.g., F, Cl, Br).

[0029] In some implementations, formula (D1) may be a part of the following formula (D1'):

[0030] In some implementations, formula (D1) may be a portion having the following formulas (D1-a) to (D1-d), but is not limited thereto:

[0031] In some embodiments, formula (D1') may be a monovalent group having the following formulas (D1'-a) to (D1'-d), but is not limited thereto:

[0032] In some embodiments, the monovalent group of formula (D2) may be a monovalent group having the following formulas (D2-a) to (D2-d), but is not limited thereto:

[0033] In some embodiments, the linker group (L) used to connect the antibody and the drug has a structure represented by the following formula (L): -C1-5 linear alkylene-(CO-NH-C1-5 linear alkylene) m -CO-, where m is an integer from 1 to 5, for example, m is 1, 2, 3, 4 or 5; and optionally one or more methylene groups in the main chain of each of the C1-5 straight-chain alkylene groups can be replaced by NH.

[0034] In some embodiments, the hydrogen in the respective backbone of the C1-5 straight-chain alkylene groups can be further converted by R. L Substituent substitution, where R L Selected from C1-5 alkyl groups, -(CH2) w -COOH and -(CH2) w -NH2, where w is an integer from 1 to 10, for example, w is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0035] In some embodiments, the linker group (L) for linking the antibody and the drug has any of the following structures, but is not limited to:

[0036] In some embodiments, the linker group (L) for linking the antibody and the drug has any of the following structures, but is not limited to:

[0037] In some embodiments, the linker group (L) for linking the antibody and the drug is further connected at any end to an oligopeptide formed of one or more amino acids (e.g., 1, 2, 3, or 4), such as oligopeptides selected from: Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)rOCH3)), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, V al-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Ci t, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly.

[0038] In some embodiments, the linking group in formula (I) is linked to Ab in formula (I) via lysine or cysteine.

[0039] In some implementations, n is an integer from 1 to 8, an integer from 1 to 6, an integer from 1 to 4, an integer from 2 to 9, an integer from 2 to 7, an integer from 2 to 5, or an integer from 3 to 6.

[0040] In some embodiments, the antibody-drug conjugate represented by formula (I) is represented by formula (Ia):

[0041] Where Ab is the antibody or antigen-binding fragment against the interleukin-6 receptor described in this article, and n is an integer from 1 to 10.

[0042] In some embodiments, the precursor of the LD portion in formula (I) is selected from XLD, where X can be selected from, but is not limited to, halogens, -NH2, -OH, and -N3. In some preferred embodiments, X is a halogen, such as Br.

[0043] In some implementations, the precursor of the LD portion in formula (I) is:

[0044] In a second aspect, the present invention provides a method for preparing the antibody-drug conjugate described herein, the method comprising:

[0045] 1) Pre-treat the antibody solution; 2) Add a reducing agent to the pre-treated antibody solution to carry out a reduction reaction; and 3) Add the precursor of the LD portion of the antibody-drug conjugate described herein to the reduced antibody solution to carry out a conjugation reaction to obtain the antibody-drug conjugate.

[0046] In a third aspect, the present invention provides the use of the antibody-drug conjugate described herein in the preparation of a medicament for treating diseases selected from arthritis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa, and juvenile idiopathic arthritis.

[0047] In a fourth aspect, the present invention provides an antibody-drug conjugate for treating diseases selected from arthritis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa, chronic kidney disease anemia, atherosclerotic cardiovascular disease, thyroid eye disease, uveitis, and juvenile idiopathic arthritis.

[0048] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate described herein, and pharmaceutically acceptable excipients.

[0049] In some embodiments, the pharmaceutical composition comprises a drug / antibody ratio (DAR) of 1-10.

[0050] In a sixth aspect, the present invention provides a kit comprising: (a) the antibody-drug conjugate or pharmaceutical composition described herein; and (b) a specification indicating the use and usage information of the antibody-drug conjugate or pharmaceutical composition.

[0051] In some implementations, the instruction manual can be a physical manual or an electronic manual.

[0052] In a sixth aspect, the present invention provides a method for treating a disease selected from arthritis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa, chronic kidney disease anemia, atherosclerotic cardiovascular disease, thyroid ophthalmopathy, uveitis, and juvenile idiopathic arthritis, comprising administering an effective amount of the antibody-drug conjugate or pharmaceutical composition described herein to a subject in need. Attached Figure Description

[0053] Figure 1 illustrates the biological activity of 206mab and its mutant.

[0054] Figure 2 shows the relative binding activities of VDJ020. Figure 2A shows the relative binding activities of 206mab and VDJ020-1 detected by ELISA, Figure 2B shows the relative binding activities of 206mab and VDJ020-10 and VDJ020-13 detected by ELISA, and Figure 2C shows the relative binding activities of 206mab and VDJ020-3 and VDJ020-14 detected by ELISA.

[0055] Figure 3 illustrates the biological activities of VDJ020. Figure 3A shows the results of detecting the biological activities of 206mab and VDJ020-1 using the 293-IL6RES reporter gene assay; Figure 3B shows the biological activities of 206mab and VDJ020-3 and VDJ020-14 using the 293-IL6RES reporter gene assay; and Figure 3C shows the biological activities of 206mab and VDJ020-10 and VDJ020-13 using the 293-IL6RES reporter gene assay.

[0056] Figure 4 shows the DSC thermal stability analysis of the VDJ020 antibody.

[0057] Figure 5 illustrates different methods for determining the biological activity of the VDJ020 antibody. Figure 5A shows the reporter gene assay for determining the biological activity of the VDJ020 antibody. Figure 5B shows the proliferation inhibition assay for determining the biological activity of the VDJ020 antibody.

[0058] Figure 6 shows a comparison of the activities of the small molecule drug (Prednisolone), the modified small molecule drug (Payload), and the modified small molecule drug linked to the linker (Payload-linker).

[0059] Figure 7 shows a comparison of the activities of the antibody (VDJ020) and the antibody-drug conjugate (VDJ009).

[0060] Figure 8 shows the curves of right ear thickness and the difference in right ear thickness over days in a mouse ear swelling animal model after different treatments. n = 6 mice, and data are expressed as mean ± SD. One-way ANOVA was used, with #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 compared to the control group; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared to the model group.

[0061] Figure 9 shows the curves illustrating the changes in the diameter of the left knee joint and the difference between the diameters of the left and right knee joints over time in a mouse model of arthritis after different treatments. n = 5, and data are expressed as mean ± SD. One-way ANOVA was used, with #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 compared to the control group; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared to the model group. Detailed Implementation

[0062] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0064] definition

[0065] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0066] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0067] The term "about" as used herein is as understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is unfamiliar with the use of this term in the context in which it is used, "about" will mean a particular value plus or minus 10%.

[0068] As used herein, "alkyl" refers to a monovalent group formed by removing one hydrogen atom from a straight-chain, straight-chain, or cycloalkanes, examples of which include methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, cyclopropyl, cyclobutyl, etc. As used herein, C1-5 alkyl refers to an alkyl group having 1-5 carbon atoms, examples of which include methyl, ethyl, propyl, butyl, and pentyl.

[0069] As used herein, "alkylene" refers to a divalent group formed by further removing a hydrogen atom from an alkyl group, preferably, the removed hydrogen atom is not on a carbon atom.

[0070] As used herein, the term "linker group" refers to the chemical moiety capable of linking the antibody described herein to a glucocorticoid. Linker groups may be readily cleaved ("cleavable linker groups"), thereby promoting the release of glucocorticoids. For example, such cleavable linker groups may be susceptible to peptidase-induced cleavage under conditions where the glucocorticoid and / or antibody remain active.

[0071] Specifically, the cleavable linker component disclosed herein comprises a peptide containing 2 to 3 amino acid residues (dipeptide or tripeptide), and more specifically, dipeptides and tripeptides selected from the group consisting of alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), and glycine-lysine (Gly-Lys). The peptide allows for cleavage of the linker group by a protease, thereby promoting the release of glucocorticoids upon exposure to intracellular proteases such as lysosomal enzymes (Doronina et al. (2003), Nature Biotechnol 21:778-784).

[0072] In this disclosure, the term "glucocorticoid" refers to a naturally occurring or synthetic steroid hormone that interacts with a glucocorticoid receptor. A "glucocorticoid group" is obtained by removing one or more hydrogen atoms from a parent glucocorticoid. The removal of hydrogen atoms facilitates the attachment of the parent glucocorticoid to a linking group. In this disclosure, hydrogen atoms are removed from any suitable -NH2, -OH group of the parent glucocorticoid. Specifically, a "glucocorticoid group" is a monovalent group obtained by removing one hydrogen atom from a parent glucocorticoid.

[0073] The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” are used interchangeably herein and refer to an amino acid polymer of any length. This polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also includes amino acid polymers that are naturally or through intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. Within this definition, polypeptides containing, for example, one or more amino acid analogs (including, for example, non-natural amino acids, etc.) and other modifications known in the art are also included. It should be understood that because polypeptides as described herein are antibody-based, they may exist as single-chain or related chains.

[0074] The term "amino acid" refers to natural, non-natural, and synthetic amino acids, including but not limited to D or L optical isomers, as well as amino acid analogs and peptide mimics. Amino acids are represented using standard single-letter or three-letter codes. "Natural L-amino acids" refers to the L optical isomers of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine ​​(C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).

[0075] In this disclosure, the term "interleukin-6 (IL-6)" refers to a multifunctional cytokine discovered in 1980, previously known as hepatocyte-stimulating factor, B-cell-stimulating factor 2, cytotoxic T-cell differentiation factor, B-cell differentiation factor, hybridoma / plasmacytoma growth factor, monocyte-granulocyte inducer type 2, and thrombopoietin. Interleukin-6 (IL-6) is produced by various cell types such as T cells, B cells, monocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells, and some tumor cells. It contains four α-helical domains with a motif of four cysteine ​​residues essential for tertiary structure. Interleukin-6 (IL-6) binds to two receptors: the specific receptor IL-6R (an 80 kDa type I transmembrane protein) and gp130, a common receptor subunit for all members of the IL-6 family of cytokines. gp130 can be expressed in all cells, but the expression of interleukin-6 receptor (IL-6R) is more restricted, and it is mainly found in hepatocytes, neutrophils, monocytes, and CD4+ cells. + T cells. Dimerization of the interleukin-6 receptor gp130 leads to the initiation of cellular events, including activation of the JAK-STAT3 pathway and ras-mediated MAP kinase signaling.

[0076] In this disclosure, the term "specific binding" refers to binding selectivity for an antigen, which can be distinguished from unwanted or nonspecific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to unrelated proteins is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. In some embodiments, the dissociation constant (Kd) of the antigen-binding molecule is ≤1 nM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

[0077] In this disclosure, the term "affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Binding affinity is typically expressed using the dissociation constant (KD), which is the sum of the dissociation rate constant and the association rate constant (kD = kJ / kE). off and k on The ratio of the rate constants is 10. Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured using conventional methods known in the art, such as surface plasmon resonance (SPR). The smaller the equilibrium dissociation constant, the more tightly the antibody or its antigen-binding fragment of this disclosure binds to IL-6R. In biological systems, good specific binders typically have a ratio of 10. -12 Up to 10 -9 The dissociation constant is within the range of [specific parameters]. In some embodiments, the antibody or its antigen-binding fragment of this disclosure has a binding affinity of approximately 10 for IL-6R. -12 10 -11 10 -10 Or 10 -9 The dissociation constant within.

[0078] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term encompasses not only complete polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chains (ScFv), mutants thereof, fusion proteins containing antibody portions (e.g., domain antibodies), and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site. Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or their subclasses), and antibodies need not be of any particular class. Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0079] In some embodiments provided herein, the antibody is a monoclonal antibody. As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. Typically, the individual antibodies constituting this population are identical, except that they may exist in small amounts due to naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method.

[0080] In some embodiments provided herein, the antibody is a humanized antibody. As used herein, a “humanized” antibody refers to a form of non-human (e.g., mouse) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the complementarity-determining region (CDR) of the receptor are replaced by residues from the CDR of a non-human species (donor antibody), such as a mouse, rat, or rabbit, having the desired specificity, affinity, and biological activity. In some cases, the Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the receptor antibody or in the introduced CDR or frame sequence, but these residues are included to further improve and optimize antibody performance. Typically, humanized antibodies will contain at least one, and usually substantially all, of two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of the human immunoglobulin common sequence. Humanized antibodies will also preferably contain at least a portion of the immunoglobulin constant region or domain (Fc), typically a portion of a human immunoglobulin. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that are altered relative to the original antibody, and are also referred to as one or more CDRs “derived” from the original antibody.

[0081] In some embodiments provided herein, the antibody is a human antibody. As used herein, "human antibody" means an antibody having an amino acid sequence corresponding to the amino acid sequence of human-produced antibodies and / or prepared using any techniques known in the art or of this invention for preparing human antibodies. This definition of a human antibody includes antibodies containing at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody containing both mouse light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art.

[0082] This disclosure also relates to amino acid sequence variants of this disclosure. When applied to proteins, a “variant” is a protein that has sequence homology with a native biologically active protein and retains at least a portion of the therapeutic and / or biological activity of the biologically active protein. Amino acid sequence variants can be prepared by introducing appropriate modifications into the nucleotide sequence of a coding molecule or by peptide synthesis. Such modifications include, for example, the deletion, insertion, and / or substitution of residues in the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain a final construct having the desired properties, such as antigen-binding activity.

[0083] As used herein, the term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in the binding of an antigen-binding molecule to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen-binding specificity. The term "variable" in this invention refers to the fact that certain segments of the variable domain are generally sequence-differentiated between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable domain. Instead, it is concentrated in three segments within the variable domains of both the light and heavy chains, called hypervariable regions (HVRs). The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are tightly held together by the FR regions and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. The constant domain does not directly participate in antibody-antigen binding but has other effector functions, such as participating in antibody-dependent cytotoxicity.

[0084] As used in this article, the term "constant region" of an antibody refers to the constant region of the antibody light chain, either alone or in combination, or the constant region of the antibody heavy chain.

[0085] As used herein, the term "hypervariant region" or "HVR" refers to a region in the variable domain region of an antibody that is highly variable in sequence and / or forms a structurally defined loop ("hypervariant loop"). Typically, a natural tetrachain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant loop and / or from the "complementarity-determining region (CDR)," which has the highest sequence variability and / or participates in antigen recognition. Those skilled in the art will understand that, unless otherwise specified, the "CDR" and "complementarity-determining region" of a given antibody or its regions (e.g., variable regions) should be understood to encompass "CDR" and "complementarity-determining region" as defined by any of the methods known in the art. It is well known to those skilled in the art that antibody CDRs can be defined using various methods, such as the Kabat definition rule based on sequence variability, the Chothia definition rule based on the location of structural loop regions, the Martin definition rule based on sequence and frame regions, the IMGT definition rule based on germline V gene amino acid sequence alignment, and reference tools for antibody humanization design based on CDR transplantation. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass any of the known schemes described above, and the amino acid sequences corresponding to the defined CDR rules should also fall within the scope of protection of this invention.

[0086] As used herein, the term "framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences typically appear in the VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0087] The term "Fc region" as used in this article is used to define the C-terminal region of the immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin typically contains two constant domains, CH2 and CH3. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the segment extending from the amino acid residue at position Cys226 or from Pro230 to its C-terminus.

[0088] As used herein, the terms “Fc receptor” and “FcR” describe receptors that bind to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are FcRs that bind IgG antibodies (γ receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. “FcR” also includes the neonatal receptor FcRn, responsible for transferring maternal IgG to the fetus.

[0089] As used herein, the term "functional Fc region" refers to having at least one effector function of a native Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require the combination of the Fc region with a binding domain (e.g., antibody variable domain) and can be assessed using various assays known in the art for evaluating such antibody effector functions.

[0090] As used herein, the term "natural sequence Fc region" includes an amino acid sequence identical to that of an Fc region found in nature. A "variant Fc region" includes an amino acid sequence that differs from the natural sequence Fc region through at least one amino acid modification, but retains at least one effector function of the natural sequence Fc region. Preferably, compared to the natural sequence Fc region or the Fc region of the parent polypeptide, the variant Fc region has at least one amino acid substitution, for example, about one to about ten amino acid substitutions in the natural sequence Fc region or the Fc region of the parent polypeptide, and more preferably about one to about five amino acid substitutions. The variant Fc region as used herein preferably has at least about 80% sequence identity with the natural sequence Fc region and / or the Fc region of the parent polypeptide, and most preferably at least about 90% sequence identity, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% sequence identity. In some embodiments of this disclosure, the Fc region of the antibody has mutations at positions 252, 254, and / or 256, wherein the amino acid residues are numbered according to the EU index in Kabat. In specific embodiments, the heavy chain of the antibody may have an amino acid sequence as shown in SEQ ID NO: 62-66 or an amino acid sequence having at least 85% sequence identity with it.

[0091] As used herein, the term "humanized antibody" comprises the splicing of amino acid residues from a nonhuman hypervariable region (HVR) and constant regions from the heavy and light chains of a human antibody to obtain the complete sequence of the humanized antibody. In some embodiments, the humanized antibody comprises at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of the nonhuman antibody, and all or substantially all of the frame regions (FRs) correspond to the FRs of the human antibody. Optionally, the humanized antibody may comprise at least a portion of the antibody constant region derived from the human antibody. An antibody in a "humanized form," such as a nonhuman antibody, refers to an antibody that has been humanized.

[0092] As used herein, the term "class" of antibody refers to the type of constant domain or constant region possessed by the heavy chain of an antibody. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively referred to as α, δ, ε, γ, and μ. In this specific embodiment, the antibody described herein is a humanized IgG1 antibody.

[0093] When applied to proteins, the term "fragment" as used herein refers to a truncated form of a naturally occurring biologically active protein that may or may not retain at least a portion of its therapeutic and / or biological activity. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with respect to a reference biologically active protein. Unless otherwise stated, "antibody fragment" and "antigen-binding fragment" are used interchangeably in the context of this invention and refer to an antibody fragment that retains the ability to specifically bind to an antigen bound by a full-length antibody, such as a fragment retaining one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as single-chain Fv (ScFv); nanobodies formed from antibody fragments; and multispecific antibodies.

[0094] In the context of polypeptides, a "linear sequence" or "sequence" is the amino acid sequence of a polypeptide from its amino to its carboxyl terminus, where adjacent residues in the sequence are continuous in the primary structure of the polypeptide. A "partial sequence" is a linear sequence of a portion of a polypeptide that is known to contain additional residues in one or two directions.

[0095] As used interchangeably in this document, "polynucleotide" or "nucleic acid" refers to a nucleotide polymer of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be conferred before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified post-polymerization, such as by conjugation with labeled components. Other types of modifications include, for example, "capping," replacing one or more naturally occurring nucleotides with analogs; internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, triphosphates, phosphoamidates, carbamates, etc.) and charged bonds (e.g., thiophosphates, dithiophosphates, etc.); modifications containing side chain portions, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); modifications containing intercalating agents (e.g., acridine, psoralen, etc.); modifications containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.); modifications containing alkylating agents; modifications with modified bonds (e.g., α-anomeric nucleic acids, etc.); and unmodified polynucleotides. Furthermore, any hydroxyl group normally present in sugars can be replaced with, for example, phosphonate groups protected by standard protecting groups, phosphate groups, or activated to prepare additional linkages with other nucleotides, or can be conjugated to a solid support. The 5' and 3' terminal OH groups may be partially phosphorylated or substituted by amine or organic end-capping groups of 1 to 20 carbon atoms. Other hydroxyl groups may also be derived as standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (such as arabinose, xylose, or lythose), pyranoses, furans, sedoheptulose, acyclic analogs, and debased nucleoside analogs such as methylnucleosides. One or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate ester is replaced by P(O)S (“thioester”), P(S)S (“dithioester”), (O)NR2 (“amide”), P(O)R, P(O)OR', CO, or CH2 (“formacetal”), wherein each R or R' is independently H or optionally contains an ether (-O-) bond, an aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl substituted or unsubstituted alkyl group (1-20 Cs). Not all links in a polynucleotide need to be identical.The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0096] As used herein, the terms “% sequence identity” or “sequence identity” are used in the context of this invention to describe the degree of similarity between two nucleotide sequences or two amino acid sequences, and have the same meaning as “percentage identity”. The percentage homology of two sequences can be calculated by dividing the number of identical residue positions by the total length of the aligned sequences after alignment, and then multiplying by 100%. Methods and tools for aligning two amino acid sequences or nucleotide sequences are well known in the art, such as the BLAST kit available on the NCBI website (Altschul, SF et al. (1990) J. Mol. Biol. 215:403-410). As used herein, “at least 85% sequence identity” means at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with that sequence.

[0097] The term "drug / antibody ratio" or "DAR" refers to the number of Ds (e.g., groups derived from small molecule glucocorticoid receptor agonists, such as glucocorticoids) linked to an Ab (e.g., an antibody). Therefore, in conjugates having the general formula Ab-(Ld)n, DAR is defined by the amount of drug loaded per antibody-drug conjugate, e.g., "n".

[0098] When referring to compounds having the formula Ab-(LD)n representing individual conjugates, the term "compound DAR" refers to the number of Ds linked to the individual Ab (e.g., drug loading or n being an integer from 1 to 10).

[0099] When referring to compounds having the formula Ab-(LD)n representing a group of conjugates, the term "group DAR" refers to the average number of D connected to Ab (e.g., drug loading or an integer or fraction of n from 1 to 10 ± 0.5, +0.4, +0.3, +0.2, +0.1).

[0100] As used herein, the term "individual" or "subject" refers to mammals, more preferably humans. Mammals also include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats, mice, and rats.

[0101] As used herein, the term "effective amount" or "therapeutic effective amount" refers to an amount of a pharmaceutical agent sufficient to produce a beneficial or desired result. Therapeutic effective amounts can vary depending on one or more of the following factors: the subject being treated and their disease condition, the subject's weight and age, the severity of the disease condition, the method of administration, etc., which can be readily determined by one of ordinary skill in the art. An effective amount of active agent can be administered in a single dose or multiple doses. Components described herein can be described as having at least an effective amount, or at least an amount effective for producing the desired result, such as a result related to a specific goal or purpose, as described herein. "Effective amount" also applies to the dose that will provide an image for detection via a suitable imaging method. A specific dose can vary depending on one or more of the following factors: the specific pharmaceutical agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the time of administration, the tissue to be imaged, and the physical delivery system carrying the agent.

[0102] As used herein, the term "pharmaceuticalally acceptable excipient" refers to an excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art. Examples include one or more of the following: carriers, excipients, adjuvants, fillers, diluents, disintegrants, lubricants, flow aids, binders, solubilizers, surfactants, emulsifiers, preservatives, antioxidants, flavoring agents, colorants, osmotic pressure regulators, and support agents.

[0103] A. Antibodies

[0104] Interleukin-6 (IL-6) is a multifunctional cytokine discovered in the 1980s, previously known as hepatocyte-stimulating factor, B-cell-stimulating factor 2, cytotoxic T-cell differentiation factor, B-cell differentiation factor, hybridoma / plasmacytoma growth factor, monocyte-granulocyte inducer type 2, and thrombopoietin. IL-6 is produced by various cell types, including T cells, B cells, monocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells, and some tumor cells. IL-6 contains four α-helical domains with motifs of four cysteine ​​residues essential for its tertiary structure. IL-6 interacts with different IL-6 receptors on the cell surface, then transmits various biological signals to different tissues and cells via downstream signaling pathways.

[0105] Interleukin-6 (IL-6) binds to two receptors: the specific receptor IL-6R (an 80 kDa type I transmembrane protein) and gp130, a common receptor subunit of IL-6 family cytokine members. gp130 can be expressed in all cells, but the expression of the interleukin-6 receptor (IL-6R) is more restricted, primarily found in hepatocytes, neutrophils, monocytes, and CD4+ cells. + T cells. Interleukin IL-6 receptor gp130 dimerization initiates cellular events including activation of the JAK-STAT3 pathway and ras-mediated MAP kinase signaling.

[0106] Studies have shown that IL-6R is highly expressed in various tumor cells, including promyelocytic leukemia, astrocytoma, glioblastoma, multiple myeloma, prostate cancer, gastric cancer, and liver cancer cells. Therefore, inhibitors targeting IL-6 or IL-6R block the binding of IL-6 to IL-6R, thereby inhibiting IL-6 pathway signaling. These IL-6 pathway inhibitors have opened a new chapter as potential therapeutic agents for various diseases.

[0107] This disclosure is based on the inventor's existing anti-human interleukin-6 receptor (IL-6R) monoclonal antibody 206mab (CN109563155A, which is incorporated herein by reference in its entirety). Through mutation modification, monoclonal antibodies with significantly improved affinity, stability and biological activity were screened out.

[0108] The sequences involved in the disclosed anti-human interleukin-6 receptor (IL-6R) monoclonal antibody are shown in Tables 1 and 2.

[0109] Table 1. Sequences of the CDR region of the 206mab and 206mab-H mutants

[0110] Table 2. Amino acid sequence of the 206mab-H variable region

[0111] Example

[0112] Preparation Example 1: Synthesis and Characterization of Compounds

[0113] I. Synthesis of intermediates

[0114] 1. Synthesis of compound 2A

[0115] Compound 2A-1 (90.0 g, 410.80 mol, 1.0 eq) was dissolved in toluene (900 mL, 10 V), and di-tert-butyl dicarbonate (116.55 g, 534.04 mol, 1.3 eq) was added. After the addition was complete, the mixture was heated to 110 °C and reacted for 16 h, with the reaction progress monitored by TLC (n-heptane:ethyl acetate = 5:1). After the reaction was complete, the mixture was cooled to 25 °C, concentrated under reduced pressure, and the resulting solid was slurried with n-hexane (3600 mL, 40 V), filtered, and dried to obtain 107.50 g of compound 2A as a white solid (HPLC purity 100.00%, yield 82%).

[0116] 1 H NMR: (400MHz, CDCl3): δ7.64–7.63(m,2H),7.50-7.48(m,1H),7.35-7.31(m,1H),6.50(br.s,1H),1.53(s,9H),1.35(s,12H).

[0117] 2. Synthesis of Compound 4A

[0118] Compound 4A-1 (50.0 g, 126.78 mol, 1.0 eq), compound 4A-2 (30.92 g, 152.14 mol, 1.2 eq), and sodium bicarbonate (21.30 g, 253.56 mol, 2.0 eq) were dissolved in 1,4-dioxane and water (1300 mL, 1:1, 26 V) and reacted at room temperature (25 °C) for 16 h. The reaction progress was monitored by TLC (dichloromethane:methanol:glacial acetic acid = 20:1:0.5). One parallel reaction was set up. After the reaction was completed, 3M HCl was added to the system to adjust the pH to 2.5. The system was extracted once with isopropyl acetate:isopropanol = 5:1 (2000 mL, 20 V), and then extracted twice with isopropyl acetate:isopropanol = 5:1 (2500 mL, 25 V). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 104.44 g of compound 4A as a white solid (LCMS purity 93.15%, yield 85%).

[0119] II. Synthesis of Compound A

[0120] Synthesis of (S)-4-(2-(2-bromoacetamyl)acetamyl)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphono)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecylhydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxane-10-yl)benzyl)phenyl)amino)5-oxopentanoic acid

[0121] a) Synthesis of Compound 2

[0122] Compound 1 (100.0 g, 510.09 mol, 1.0 eq) was dissolved in toluene (2000 mL, 20 V), cooled to 0 °C in an ice bath, and then diisobutylaluminum hydride (108.82 g, 765.14 mol, 1.5 eq) was slowly added dropwise. After the addition was complete, the reaction was carried out at room temperature (25 °C) for 3 h, and the reaction progress was monitored by TLC (n-heptane:ethyl acetate = 1:1). After the reaction was complete, 10% hydrochloric acid (2500 mL, 25 V) was added dropwise to the system. The system was filtered with diatomaceous earth, separated, and the aqueous phase was extracted three times with DCM (800 mL, 8 V). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 88.08 g of compound 2 as a white solid. 1 H NMR content 72.90%, yield 87%.

[0123] 1 H NMR (400MHz, DMSO-d6): δ9.98 (s, 1H), 7.88 (d, J = 8.0Hz, 2H), 7.64 (d, J = 8.0Hz, 2H), 4.75 (s, 2H).

[0124] b) Synthesis of Compound 3

[0125] Compound 2 (76.75 g, 385.59 mmol, 1.5 eq), compound 2A (82 g, 257.06 mmol, 1.0 eq), and potassium carbonate (177.52 g, 1.29 mol, 5.0 eq) were dissolved in anhydrous tetrahydrofuran (1640 mL, 20 V). The mixture was deoxygenated for 10 min, purged with nitrogen three times, and then a 1,1-bis(diphenylphosphine)ferrocene palladium dichloromethane complex (10.50 g, 12.85 mmol, 0.05 eq) was added. The mixture was deoxygenated for 10 min, purged with nitrogen three times, and the reaction was carried out at 100 °C for 16 h. The reaction progress was monitored by TLC (n-heptane:ethyl acetate = 10:1). The system was filtered through diatomaceous earth, concentrated under reduced pressure, and purified by silica gel column chromatography (n-heptane:ethyl acetate = 20:1 to 10:1) to give compound 3 as a white solid, 32.00 g. 1 H NMR content 93.92%, yield 40%.

[0126] 1 H NMR (400MHz, CDCl3): δ9.94(s,1H),7.76(d,J=8.0Hz,2H),7.32(d,J=8.0Hz,2H),7.18(s,2H),6.82(s,1H),6.64(s,1H),3.98(s,2H),1.49(s,9H).

[0127] c) Synthesis of Compound 4

[0128] Compound 3 (32.00 g, 102.77 mmol, 1.0 eq) and compound 3A (38.69 g, 102.77 mmol, 1.0 eq) were dissolved in acetonitrile (320 mL, 10 V), cooled to 0 °C in an ice bath, and then perchloric acid (6.45 g, 64.23 mmol, 5 eq) was slowly added dropwise. After the addition was complete, the mixture was warmed to 25 °C and reacted for 2 h. The reaction progress was monitored by TLC (n-heptane:ethyl acetate = 1:4). After the reaction was completed, a saturated sodium bicarbonate solution (800 mL, 25 V) was added dropwise to the system, followed by DCM (800 mL, 25 V). The mixture was separated, and the aqueous phase was extracted twice with DCM (800 mL, 25 V). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (n-heptane: ethyl acetate = 1:1 to 1:4) to give 30.30 g of compound 4 as a yellow solid (LCMS purity: 59.63%, yield 52%).

[0129] d) Synthesis of compound 5

[0130] Compound 4 (29.00 g, 50.91 mmol, 1.0 eq) and compound 4A (41.76 g, 86.54 mmol, 1.7 eq) were added to N,N-dimethylformamide (203 mL, 7V), followed by N,N-diisopropylethylamine (19.74 g, 152.73 mmol, 3.0 eq). The mixture was cooled to 0°C in an ice bath, and a 50% DMF solution of T3P (32.39 g, 101.81 mmol, 2.0 eq) was slowly added dropwise. The mixture was stirred at 25°C for 16 h. The reaction was monitored by TLC (n-heptane:ethyl acetate = 1:4). After the reaction was complete, water (145 mL, 5 V) was added, followed by ethyl acetate (435 mL, 15 V). The mixture was extracted twice, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (n-heptane:ethyl acetate = 1:1 to 1:4) to give 44.5 g of crude compound 5 as a yellow solid.

[0131] The crude product was dissolved in DMF and purified by reverse Pre-HPLC (0.1% ammonium bicarbonate / water / acetonitrile). The fraction was lyophilized to give 12.20 g of compound 5 as a pale yellow solid (LCMS purity 97.66%, yield: 23%).

[0132] e) Synthesis of Compound 6

[0133] Compound 5 (500.00 mg, 483.47 μmol, 1.0 eq) was dissolved in N,N-dimethylformamide (3.20 mL, 6.25 V), and tetrazolium (338.68 mg, 4.84 mmol, 10.0 eq) and N,N-diethylphosphite di-tert-butyl ester (1.45 g, 5.80 mmol, 12.0 eq) were added. After addition, the mixture was stirred at 25 °C for 2.5 h, and the reaction progress was monitored by TLC (n-heptane:ethyl acetate = 1:4). The reaction of compound 5 was considered complete. The mixture was cooled to 0 °C in an ice bath, and 35% hydrogen peroxide (258.42 mg, 2.66 mmol, 5.5 eq) was slowly added dropwise. After addition, the mixture was stirred at 25 °C for 1 h, and the reaction progress was monitored by TLC (n-heptane:ethyl acetate = 1:4). Eleven parallel reactions were then set up. After the reaction was complete, 5% sodium thiosulfate solution (188 mL, 5 V) was added, followed by ethyl acetate (564 mL, 15 V). The mixture was extracted twice, and the organic phases were combined. The organic phases were concentrated under reduced pressure, and then n-heptane (188 mL, 5 V) was added and stirred. The mixture was filtered to obtain 6.80 g of compound 6 as a pale yellow solid (LCMS purity 98.62%, yield: 85%).

[0134] f) Synthesis of Compound 7

[0135] Compound 6 (480 mg, 0.39 mmol, 1.0 eq) was dissolved in acetonitrile (3 mL, 6.25 V), and piperidine (0.32 mL, 3.315 mmol, 8.5 eq) was added. The mixture was stirred at room temperature (25 °C) for 25 min. HPLC analysis confirmed the reaction was complete. The reaction solution was evaporated to dryness under reduced pressure. The residue was repeatedly slurried with petroleum ether for 1 h each time until impurities were removed. Filtration yielded 390 mg of compound 7 as a white solid (HPLC purity 78.38%, yield: 95%).

[0136] g) Synthesis of Compound 8

[0137] Bromoacetic acid (108.3 mg, 0.78 mmol, 2.0 eq) was dissolved in DMF (3 mL, 7.7 V), and EEDQ (192.89 mg, 0.78 mmol, 2.0 eq) was added. The mixture was stirred at 25 °C for 1 h. A DMF solution of compound 7 (390 mg, 0.39 mmol, 1.0 eq) (3 mL, 7.7 V) was added to the reaction solution, and the reaction was continued for 2.5 h. HPLC analysis confirmed the reaction was complete. The reaction solution was diluted with DCM (200 mL), washed with 5% phosphoric acid solution (160 mL * 2), NaHCO3 solution (100 mL * 2), and saturated brine. The organic phase was dried, filtered, and the filtrate was evaporated under reduced pressure to obtain 440 mg of a pale yellow oily substance of compound 8 (HPLC purity 88.18%, yield: 98%).

[0138] h) Synthesis of compound A

[0139] Compound 8 (293 mg, 0.26 mmol) was dissolved in DCM (10 mL), and TFA (5 mL) was added. The mixture was stirred at room temperature for 45 min. The reaction was confirmed to be complete by HPLC. The reaction solution was evaporated to dryness under reduced pressure, and the residue was purified by preparative HPLC to obtain 120 mg of compound A as a white solid powder (HPLC purity 97.69%, yield: 48%).

[0140] LCMS: 956.2 [M+H+];

[0141] 1H NMR: (400MHz, DMSO-d6): δ9.88(s,1H),8.52(t,J=6.0Hz,1H),8.23(d,J=7.6Hz,1H),7.39–7.23(m,8 H),6.92(d,J=7.6Hz,1H),6.16(dd,J=10.0,1.6Hz,1H),5.93(s,1H),5.48(s,1H),4.92-4.91(m,3H), 4.59-4.53(m,1H),4.40-4.38(m,1H),4.30(s,1H),3.94(s,2H),3.90(s,2H),3.80(q,J=2.8Hz,2H),2 .27-2.25(m,2H),2.24-1.88(m,4H),1.80-1.49(m,7H),1.42(s,3H),1.06-1.03(m,2H),0.82(s,3H).

[0142] Preparation Example 2: Preparation of Antibody-Drug Conjugates

[0143] 1. Antibody pretreatment

[0144] Add Na2HPO4 (40mM) to the antibody solution described in this article, adjust the pH to 7.0, and then add EDTA stock solution to make the final system contain 2mM EDTA.

[0145] 2. Preparation of antibody-drug conjugates

[0146] TCEP solution (10 mM, 1.8–2.2 eq) and PB buffer were added to the antibody solution (10 mg / mL, PB system, pH 7.0), mixed thoroughly, and reacted at 22°C for 3 h. Then, Linker-payload solution (10 mg / mL, 40 mM PB–2 mM EDTA system, compound A described herein) was added to the reaction solution, and reacted at 22°C for 2 h. Free compound A was removed using a 30 kDa ultrafiltration membrane, and the buffer system was replaced with PBS buffer. Finally, the formulation components were added to the ultrafiltration collection solution in proportion to complete the preparation of the antibody-drug conjugate (final system PBS buffer, containing 8% sucrose and 0.04% Tween 80). The obtained antibody-drug conjugate was stored at -80°C for later use.

[0147] Example 1. Expression and purification of 206mab-mut in HEK293 cells

[0148] One day before transfection, 50 mL of KOP293 cells (Zhuhai Kairui) were prepared at a density of 1.2 × 10⁻⁶ cells. 6HEK293 cells per mL were cultured in 250 mL Erlenmeyer flasks at 135 rpm, 37 °C, and 5% CO2 for 20–25 h until the cell density reached 2 × 10⁶ cells / mL. 6 Cells with a density of approximately [number] cells / mL, in logarithmic growth phase, and a viability of over 95% were used for transfection experiments. The antibody's heavy and light chain encoded nucleic acids (sequences shown in SEQ ID NO: 67-83) were cloned into PEE6.4 and PEE14.4 vectors respectively via HindIII and EcoRI restriction sites to construct recombinant plasmids. 50 μg of recombinant plasmids expressing 206mab and the 206mab mutant 206mab-mut-1-17 (amino acid sequences shown in Table 3) were added to Opti-MEM, bringing the total volume to 2 mL. The mixture was gently mixed, and 250 μg of PEI was added to Opti-MEM, bringing the total volume to 2 mL. The mixture was incubated at room temperature for 5 min. 2 mL of PEI dilution buffer was added to the corresponding plasmid dilution buffer, mixed, and incubated at room temperature for 20 min. Gently rotate and shake the Erlenmeyer flask containing HEK293 cells, slowly add PEI-DNA mixture, and then incubate the transfected cells in a shaker at 135 rpm, 37°C, and 5% CO2. After 24 h of transfection, add 1 mL of 50×KT-Feed and 100 μL of 500×VPA. After 5 days of transfection, centrifuge at 3000 rpm for 15 min to collect the cell culture medium. After high-speed centrifugation of the cell expression supernatant, filter to remove impurities, and perform affinity chromatography on a HiTrap MabSelect SuRe pre-packed column. After rinsing the column with pure water, it was equilibrated with PB buffer. The supernatant was then loaded onto the chromatography column for binding. After loading, the column was rinsed with PB buffer to the baseline. The protein was then eluted with 0.1M citrate elution buffer. The eluted protein was neutralized with 1M Tris-HCl, concentrated appropriately, and loaded onto a PBS-equilibrated Superdex 200 for further purification. The collected protein was concentrated and identified by SDS-PAGE electrophoresis and SEC-HPLC purity detection. The purity results are shown in Table 4.

[0149] Table 3. Amino acid sequence of the 206mab-H mutant

[0150] The amino acid sequences of the light chain variable region of 206mab and its mutant 206mab-mut-1 to 17 are as follows:

[0151] DIQMTQSPSSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCGQGNRLPYTFGQGTKVEIK(SEQ ID NO: 60)

[0152] The light chain amino acid sequences of 206mab and its mutant 206mab-mut-1 to 17 are as follows:

[0153] DIQMTQSPSSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCGQGNRLPYTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 61)

[0154] Table 4. SEC purity results for 206 mab-mut

[0155] Example 2. Affinity assay of 206mab-mut antibody expressed transiently.

[0156] The affinity of 206mab-mut-1–17 and its parent antibody 206mab for IL6R was determined using bio-layer interferometry (BLI). All 18 monoclonal antibodies were diluted to 20 nM, and human IL6R protein (P08887-1) was serially diluted from 50 nM to approximately 3.125 nM. The 18 monoclonal antibodies were loaded onto ProA probes at 400 rpm / min, and then different gradients of diluted hIL6R were bound to the antibody-attached ProA probes. The probes containing the antigen-antibody complexes were placed in a Q buffer free of analyte to allow dissociation of the bound analyte. The probes were then immersed in a regeneration buffer to remove residual bound analyte, regenerating and storing the probes. Affinity data were obtained by integrating the fitted lines at each concentration using the Gator software; the results are shown in Table 5.

[0157] Table 5. Affinity results for 206mab and 206mab-mut-1 to 17

[0158] Example 3. Determination of biological activity of 206mab-mut antibody by proliferation inhibition assay

[0159] DS-1 cells (CRL-11102) were starved for 24 h, and then populated in 96-well plates at a density of 2 × 10⁻⁶ cells / well. 4 80 μl / well was used for plating. IL-6 was diluted to 20 ng / mL with 1640 medium (Gibco) containing 10% FBS, and 10 μl / well was added to the sample wells and positive control wells. 10 μl / well of 1640 medium containing 10% FBS was added to the negative control wells. 206mab and 206mab-mut-1–17 antibodies were diluted to 1 mg / mL with 1640 medium, and then serially diluted 4-fold to 8 concentrations. 10 μl / well was added to the corresponding wells of a 96-well plate, with two replicates for each concentration. 10 μl / well of 1640 medium was added to the positive and negative control wells. After thorough mixing with the added samples, the plate was incubated at 37°C for 72 h. After melting and mixing the MTS assay reagent (Promega), add 20 μl / well to each of the 96 wells of the plate. After mixing by shaking in a plate shaker, incubate at 37°C for 0.5 h. Read the values ​​at 490 nm using a TECAN multi-mode microplate reader. Plot the data using Prism 5 (GraphPad) software and calculate the relative biological activity of 206 mab mut using the formula: (206 mab IC50) 50 / 206mab mut IC 50 *100%, the biological activity of 206mab was set to 100%, and the results are shown in Table 6 and Figure 1. The biological activities of the three antibody molecules 206mab-mut-3, 206mab-mut-10, and 206mab-mut-14 were significantly improved compared with 206mab.

[0160] Table 6. Biological activity results of 206mab-mut

[0161] Example 4. Detection of the relative binding activity of VDJ020

[0162] Five antibodies, 206mab-mut-1, 206mab-mut-3, 206mab-mut-10, 206mab-mut-13, and 206mab-mut-14, were selected. Each antibody underwent modification of three amino acid sites in the Fc region of the heavy chain (M252Y / S254T / T256E) to extend the antibody half-life. The modified antibodies were named VDJ020-1, VDJ020-3, VDJ020-10, VDJ020-13, and VDJ020-14, respectively, and their sequences are shown in Table 7.

[0163] Table 7. Antibody heavy chain sequences after Fc region modification

[0164] The five antibody heavy and light chain genes were cloned into the pCDNA3.4 vector via HindIII & EcoRI double restriction sites. The heavy and light chains were co-transformed into HEK293, and expressed and purified according to the method in Example 1 to obtain five antibodies: VDJ020-1, VDJ020-3, VDJ020-10, VDJ020-13, and VDJ020-14. Relative binding activity was detected using ELISA. Human IL6R was diluted to 1 μg / mL, 100 μL / well, and coated overnight at 4°C. The next day, the plates were washed and blocked with 5% skim milk for 2 hours. Then, the plates were washed again and antibody samples of different concentrations were added, and incubated at 37°C for 1 hour. After washing, 100 μL of HRP-labeled goat anti-human IgG secondary antibody was added to each plate, and incubated at 37°C for 30 minutes. Add TMB chromogenic solution, incubate at 37°C for 10 minutes, and finally add 50 μL H2SO4 to stop the reaction. Read the OD value using a TECAN multi-functional microplate reader. 450 The values ​​were calculated, and Prism 5 (GraphPad) software was used to plot the data and calculate the relative binding activity using the formula: (206 mab EC 50 / VDJ020EC 50 The binding activity of 206 mab was set to 100% (*100%), and the results are shown in Figure 2 and Table 8. The relative binding activities were basically consistent.

[0165] Table 8. Relative binding activity results of VDJ020

[0166] Example 5. Affinity determination of VDJ020

[0167] The affinity of five antibodies (VDJ020-1, VDJ020-3, VDJ020-10, VDJ020-13, and VDJ020-14) for IL6R was detected using the SPR method and a Biacore instrument. The results are shown in Table 9.

[0168] Table 9. Affinity test results of VDJ020

[0169] Example 6. Reporter gene assay for the biological activity of VDJ020

[0170] A plasmid containing the NF-κB Luc2p Hygromycin (Promega) gene was transduced into HEK293 cells (ATCC) to construct the HEK293-NF-κB luciferase reporter gene viability test cell line. HEK293-NF-κB reporter gene cells were digested with trypsin at room temperature for approximately 1 min. Digestion was stopped by adding DMEM (Gibco) medium when cells detached. The cells were gently mixed and centrifuged at 200–300 g for 5 min. The supernatant was discarded, the cells were resuspended, and the cells were counted. The cell density was adjusted to 5 × 10⁶ cells / year. 5 Cells / mL were added to the corresponding wells at a rate of 80 μL / well. IL6 was diluted to 50 ng / mL and added to the sample and positive control wells at 10 μL / well. 10 μL of basal medium was added to the negative control wells. 206 mab and VDJ020 antibodies were diluted to 200 μg / mL with culture medium and serially diluted 5-fold to 8 concentrations. 10 μL of each concentration was added to the corresponding wells of a 96-well plate, with two replicates for each concentration. 10 μL of basal medium was added to the positive and negative control wells. The cell culture plates were incubated at 37°C in a 5% CO2 cell culture incubator for 22 h. The next day, 100 μL of the melted and mixed One Lite assay reagent, which had been brought to room temperature, was added to the above 96-well plates. The plates were shaken and mixed for 3 min, and the chemiluminescence values ​​were read using a multi-mode microplate reader. The data were plotted using Prism 5 (GraphPad) software, and the relative biological activity of VDJ020 was calculated using the formula: (206 mab IC 50 / VDJ020IC 50 *100%, with the biological activity of 206 mab set at 100%. Results are shown in Table 10 and Figure 3. The data indicate that the biological activity of VDJ020-14 is 2.6 times higher than that of 206 mab.

[0171] Table 10. Biological activity results of VDJ020

[0172] Example 7. Detection of the binding ability of VDJ020 and FcRn

[0173] The affinity of VDJ020 for FcRn was determined under different pH buffer systems. Human RcRn (hFcRn, Acrobiosystems) was labeled with a biotinylated kit, diluted to a concentration of 25 nM, loaded onto an SA probe, and then reacted with different gradients of VDJ020 antibody in buffer systems at pH 6.0 and pH 7.4, respectively. The antibodies were then dissociated in the corresponding pH buffers, and the affinity values ​​were obtained by fitting the data using the instrument's built-in software. The results are shown in Table 11. The results showed that the binding of VDJ020 antibody to hFcRn was significantly improved compared to 206 mab in both pH 6.0 and pH 7.4 buffer systems.

[0174] Table 11. Affinity results of VDJ020 antibody and hFcRn

[0175] Example 8. Analysis of the thermostability of VDJ020 antibody

[0176] The thermal stability of humanized IgG1 antibodies was analyzed by the Institute of Process Engineering, Chinese Academy of Sciences, using a MicrolCal™ VP-DSC system (Malvern, UK) based on differential scanning calorimetry (DSC). Samples and buffer solutions were degassed by a vacuum pump for 5 minutes before measurement. Instrument parameters were as follows: heating rate 60℃ / h; temperature range 25-95℃; feedback mode: none. Sample spectra were obtained after subtracting the buffer solution. Data analysis was performed using the built-in MicrolCal Origin 7.0 (Origin-Lab Corp., MA) software. Results are shown in Table 12 and Figure 4. Tm1 is the melting temperature at the interface between the two CH2 domains, Tm2 is the melting temperature at the interface between the heavy and light chains, and Tm3 is the melting temperature at the interface between the two CH3 domains. The data show that the Tm1 value of the VDJ020 antibody decreased slightly, while Tm2 and Tm3 remained essentially unchanged.

[0177] Table 12. Results of the thermostability of VDJ020 antibody

[0178] Example 9. Determination of the isoelectric point of VDJ020

[0179] The isoelectric point (pI) of VDJ020 was determined using a Maurice dual-function analysis system based on capillary electrophoresis. A polymer was coated on the inner wall of the capillary to reduce electroosmotic flow. The sample and amphoteric electrolyte were mixed and injected. Acid and alkali solutions were added to the two electrode cells, respectively. After applying voltage, a pH gradient gradually formed in the capillary electrolyte solution. When each solute migrated to its respective pI in the capillary, it became neutral, forming a focused zone. Whole-column imaging was then used for detection. The results are shown in Table 13. The isoelectric point of VDJ020 did not show a significant change compared to 206 mAb.

[0180] Table 13. Isoelectric point test results of VDJ020

[0181] Example 10. Stability detection of VDJ020 antibody

[0182] Based on all the above data, two antibodies, VDJ020-3 and VDH020-14, were selected for stability testing, with two concentrations of 67 mg / mL and 2 mg / mL for each antibody. High-concentration samples were subjected to accelerated treatment at 40℃ for 0, 1, 2, and 4 weeks; low-concentration samples were aliquoted into two groups and subjected to repeated freeze-thaw cycles of 3, 5, and 10 times, and accelerated treatment at 40℃ for 0, 1, 2, and 4 weeks, respectively. The prepared samples were evaluated for protein content, purity, isoelectric point, charge heterogeneity, reduced and non-reduced CE-SDS, biological activity, and relative binding activity to assess the stability of the VDJ020 antibodies. The results are shown in Tables 14–16. All data indicate that both VDJ020-3 and VDJ020-14 molecules are relatively stable under both accelerated high-temperature conditions at 40℃ and freeze-thaw conditions.

[0183] Table 14. High-temperature stability results of the 67 mg / mL formulation

[0184] Table 15. High-temperature stability results of the 2 mg / mL formulation

[0185] Table 16. Freeze-thaw stability results of the 2 mg / mL formulation

[0186] Example 11. Detection of the biological activity of VDJ020 antibody using different methods

[0187] VDJ020-3 and VDJ020-14 were detected using the reporter gene assay and proliferation inhibition assay, respectively, following the same methods as above. The results are shown in Table 17 and Figure 5. The data indicate that the biological activity of VDJ020-14 detected by the reporter gene assay was 2.8 times higher than that of VDJ020-3. The biological activity of VDJ020-14 detected by the proliferation inhibition assay was 3.2 times higher than that of VDJ020-3. The biological activity of VDJ020-14 was approximately 3 times higher than that of VDJ020-3.

[0188] Table 17. Results of detection of VDJ020 antibody biological activity by different methods

[0189] Example 12: Detection of Small Molecule Biological Activity

[0190] In this embodiment, the biological activity of small molecules in antibody-drug conjugates was detected using the IL6R-OE MMTV Luc2p HEK293 34# cell / reporter gene assay.

[0191] 1. Materials and Instruments

[0192] 1.1 Main Instruments

[0193] 1.2 Sample Information

[0194] 1.3 Reagents and Consumables

[0195] 2. Experimental Procedure

[0196] 2.1 The culture medium suitable for IL6R-OE MMTV Luc2p HEK293 34# passage: DMEM, 10% FBS, 1% PS, 200 μg / ml hygromycin B, 600 ug / ml G418.

[0197] 2.2 Prepare HEK293 medium: DMEM, 10% FBS, 1% PS.

[0198] 3.3 Trypsin digestion of IL6R-OE MMTV Luc2p HEK293 34#. After neutralization in HEK293 medium, centrifuge at 900 rpm and discard the supernatant. Resuspend the cells in HEK293 medium. Add 40,000 / 135 μL of the cell suspension to each well of a 96-well plate and incubate for 3-4 hours until adherence.

[0199] 3.4 Prepare 10×prednisolone serial dilution working solutions (ug / ml) using HEK293 medium according to Table 18.

[0200] Table 18: Prednisolone graded dilution working solutions

[0201] 3.5 Prepare 10X VDJ009-Payload gradient dilution working solution (nM) using HEK293 medium according to Table 19.

[0202] Table 19: Concentration of VDJ009-Payload Gradient Dilution Working Solution

[0203] 3.6 Prepare 10×VDJ009-Payload-linker serially diluted working solution (nM) using HEK293 medium according to Table 20.

[0204] Table 20: Concentration of VDJ009-Payload-linker Gradient Dilution Working Solution

[0205] 3.7 Add 15 μL of each graded dilution of the working solution to the corresponding cells and incubate at 37°C and 5% CO2 for 16 hours.

[0206] 3.8 After incubation, bring the cell plate to room temperature; add 100 μl of One lite luciferase assay reagent in the dark, incubate at room temperature for 5 minutes, and then read the chemiluminescence signal value.

[0207] 4. Results Analysis

[0208] As shown in Figure 6, the experimental data indicate that the activity of the modified small molecule drug (Prednisolone) (Payload) is significantly increased (approximately 70-fold), and the activity does not change significantly after the Payload binds to the linker. This is beneficial for reducing the dosage, improving patient tolerability, and reducing adverse reactions.

[0209] Example 13: Detection of antibody biological activity in VDJ009

[0210] In this embodiment, the biological activity of the antibody fraction in VDJ009 was detected using the 293-IL6Res cell / reporter gene assay.

[0211] 1. Materials and Equipment

[0212] 1.1 Reagents and Materials

[0213] The reagents and materials used in this embodiment are as follows:

[0214] DMEM (Gibco), FBS (Gibco), 0.25% Trypsin-EDTA (1X) (Gibco), Recombinant Human IL-6 Protein (R&D), Hygromycin (Bai Rui Ji), One Lite Assay Reagent (Vazyme), Pen Strep (Gibco), 1×DPBS (CORNING), 96-well Dilution Plate (NEST), 96-well White-walled Transparent Plate (Costar).

[0215] 1.2 Main Equipment and Consumables

[0216] The equipment used in this embodiment is shown below:

[0217] CO2 incubator (Thermo), multi-functional microplate reader (TECAN), vortex shaker (Qilinbell).

[0218] 2. Experimental Procedure

[0219] 2.1 Preparation of cell culture medium

[0220] Basic culture medium: Measure 445ml of DMEM medium, add 50ml of FBS and 5ml of PS, mix thoroughly, and store at 4℃.

[0221] Complete culture medium: Add 200 μl of hygromycin to 100 ml of basal culture medium, so that the final concentration of hygromycin is 100 μg / ml.

[0222] 2.2 IL6 dilution

[0223] Dilute the dry powder IL6 to 100 μg / ml with basal culture medium, then dilute it 2000 times to a final concentration of 50 ng / ml. Aliquot into 1 ml vials and store at -80℃. Shelf life is 1 year.

[0224] 2.3 Cell Culture

[0225] 293-IL6Res cells were revived using basal medium. The medium was changed the next day, the culture medium was discarded, and the cells were washed once with DPBS. Fresh basal medium was added, and the cells were passaged on the third day (passage 1) using basal medium. Passages 2 and subsequent passages were cultured in complete medium, and biological activity was measured using cells from passage 3 and subsequent passages. When the cells covered 70–90% of the bottom area of ​​the cell culture flask, passage and viability testing could be performed. The optimal period for viability testing was 7–30 days after revival (passage 3 and subsequent passages).

[0226] 2.4 Assay for biological activity

[0227] 2.4.1 Cell Treatment

[0228] Rinse cells with preheated sterile PBS for 3–5 seconds, then aspirate the PBS. Add trypsin and react at room temperature for about 1 minute. When cells detach, add basal culture medium to stop digestion. Gently mix by pipetting, then centrifuge at 200–300g for 5 minutes. Discard the supernatant, resuspend and count the cells, adjusting the cell density to 5 × 10⁶ cells / year. 5 Add 80 μl per well to each well, with a cell / ml ratio.

[0229] 2.4.2 IL6 dissolution

[0230] Take out the IL6 stored at -80℃, thaw it at room temperature, invert and mix it, and add 10 μl / well of basal culture medium to the sample wells and positive control wells. Add 10 μl / well of basal culture medium to the negative wells.

[0231] 2.4.3 Sample Dilution

[0232] The reference and test samples were diluted to 20 μg / ml with basal culture medium, and then serially diluted 5-fold to obtain 9 concentrations. 10 μl / well was added to the corresponding wells of a 96-well plate, with two replicates for each concentration. 10 μl / well of basal culture medium was added to both the positive and negative control groups. The cell culture plates were incubated at 37°C in a 5% CO2 incubator for 22 h.

[0233] 2.4.4 Readings

[0234] Add 100 μl / well of the melted and mixed One Lite assay reagent, which has been brought to room temperature, to the above 96-well plate. Shake well for 3 min, let stand for 3–5 min, and read the chemiluminescence value (RLU) using a multi-mode microplate reader.

[0235] 3. Result Calculation

[0236] 3.1 Result Determination

[0237] The experimental results are valid only if the following parameters are met:

[0238] 3.1.1 The four-parameter curve approximates an inverted "S" shape.

[0239] 3.1.2 Correlation coefficient of four-parameter curves (R²) 2 The value is greater than 0.95.

[0240] 3.1.3 The number of points on the upper platform shall not be less than 2, and the number of points on the lower platform shall not be less than 2.

[0241] 3.2 A four-parameter fitting curve was used to process the data. The logarithm of the concentration of the standard or test sample was plotted on the x-axis, and the RLU value was plotted on the y-axis. The IC50 values ​​of the standard and test sample were calculated. 50 Value. Calculated using the following formula:

[0242] 4. Results Analysis

[0243] As shown in Figure 7, VDJ009 is the antibody-drug conjugate described in this invention, and VDJ020 is the antibody used in the antibody-drug conjugate described in this invention. Data shows that the IC50 values ​​of VDJ009 and VDJ020 did not change significantly, indicating that the antibody fraction of VDJ009 has good activity and does not affect the drug's targeting effect.

[0244] Example 14: Efficacy experiment of VDJ009 on FITC-induced hIL6 / hIL6R mouse ear swelling animal model

[0245] The reagent information used in this embodiment is shown below:

[0246] Injectable saline solution (Shijiazhuang No. 4 Pharmaceutical Co., Ltd.), fluorescein isothiocyanate (SIGMA), dibutyl phthalate (SIGMA), acetone (Thermo).

[0247] 1. Sample preparation

[0248] Preparation of antibody (VDJ020):

[0249] Take a certain amount of antibody (VDJ020) and dilute it with physiological saline for injection to a suitable concentration.

[0250] Preparation of antibody-drug conjugate (VDJ009):

[0251] A certain amount of antibody-drug conjugate (VDJ009) was taken and diluted with physiological saline for injection to a suitable concentration as the high-dose group. Then it was diluted 3 times with physiological saline as the low-dose group.

[0252] 2. Preparation of molding agent

[0253] Preparation of 1.5% FITC:

[0254] Solvent: Acetone / butyl phthalate mixed at a 1:1 volume ratio (operated in a fume hood);

[0255] 1.5% FITC: Weigh the amount of FITC needed for one sensitization, protected from light. Mix the FITC powder and the solvent mentioned above evenly in a ratio of 1.5:100 to obtain a 1.5% FITC sensitizer. The FITC solution should be prepared fresh for use, protected from light, and shielded from light with aluminum foil.

[0256] Sensitization period: 2800ul solvent for group G1, 2800ul FITC for groups G2-G5, prepare 12ml (0.18g FITC) modeling agent, each group is packaged in 3000ul.

[0257] Excitation phase: 150ul of solvent for group G1, 150ul of FITC for groups G2-G5, prepare 1ml (0.015g FITC) of modeling agent, and dispense 250ul of each group.

[0258] 3. Experimental System

[0259] 3.1 Laboratory Animals

[0260] 3.2 Feeding and Management

[0261] Laboratory animals were housed in an SPF-grade animal barrier facility. The animals underwent a 3-day acclimatization period before the experiment.

[0262] The ambient temperature and humidity of the barrier facility are controlled within the following ranges:

[0263] Temperature: 20-26℃

[0264] Humidity: 40-70%

[0265] Lighting: Alternates every 12 hours

[0266] Rat cages: 3-4 animals per cage, with corn cob bedding sterilized and cleaned, and changed weekly. Each cage has a label indicating the number of animals, sex, strain, receipt time, group, and trial start time.

[0267] Feed and water: SPF grade growth and reproduction feed. Drinking water is sterile purified water. Animals have free access to sterile food and water.

[0268] Animal number: Apply to toes or tail

[0269] 4. Experimental Design

[0270] 4.1 Modeling and Grouping

[0271] Adaptation period: Before the start of the experiment, the test animals undergo a 3-day adaptation period at the test site.

[0272] Grouping: After the adaptation period, students were randomly assigned to groups based on weight, ear thickness, and ear condition (abnormal ears were removed). Before the abdominal sensitization test, the abdomen of each group was shaved (area: 3.5cm × 3.5cm), and this was recorded as day 0 (D0).

[0273] Modeling steps: During the abdominal sensitization period, on day 0, the modeling agent was applied evenly to the mouse abdomen (380 μL of solvent for the blank group and 380 μL of 1.5% FITC for the model group). During the challenge period, on day 7, the agent was applied evenly to the right ear of the mouse (20 μL of solvent for the blank group and 20 μL of 1.5% FITC for the model group). After application, the mouse was restrained for 1-2 minutes to allow for full absorption of the agent.

[0274] The specific dosages for each group are shown in Table 21 below:

[0275] Table 21: Dosage and administration regimen

[0276] Groups G1–G6 were given the drug 2 days before stimulation (D5), for a total of one administration.

[0277] 4.2 Detection Indicators

[0278] 4.2.1 Clinical observation of animals: Observation frequency and time for all animals: Observe twice a day during the experiment (with an interval of at least 6 hours between morning and afternoon).

[0279] Observation content: including death and clinical manifestations.

[0280] 4.2.2 Animals for weight measurement: All animals

[0281] Detection times: D0, D2, D4, D6, D8, D9, D10, D11, D12, D13.

[0282] 4.2.3 Detection of ear swelling

[0283] Before sensitization at D0, before stimulation at D7, and on D8, D9, D10, D11, D12, and D13, the thickness of the right ear was measured with vernier calipers and recorded. This was repeated three times in parallel (by the same person).

[0284] 4.3 Test Results

[0285] As shown in Figure 8, after D7 stimulation, VDJ009 at 1.14 mg / kg exhibited an inhibitory effect, while VDJ009 at 3.42 mg / kg and VDJ020 at 10 mg / kg showed similar inhibitory effects. VDJ009 at 10.26 mg / kg exhibited a stronger immunosuppressive effect, indicating that VDJ009 only requires one-third the dose of VDJ020 to achieve the same therapeutic effect. These results suggest that the antibody-drug conjugate of the present invention is beneficial for reducing dosage, improving patient tolerability, and reducing adverse reactions.

[0286] Example 15: Efficacy experiment of VDJ009 in an IL-6 / mBSA-induced hIL6 / hIL6R mouse arthritis model. Information on some of the reagents used in this example is shown below:

[0287] Injectable saline (Shijiazhuang No.4 Pharmaceutical), mBSA (SIGMA), hIL-6 (Beijing Weidejie).

[0288] 1. Reagent preparation

[0289] 1.1 Sample Preparation

[0290] VDJ020 configuration:

[0291] Take a certain amount of VDJ020 and dilute it with physiological saline for injection to a suitable concentration. Prepare it fresh each time you use it.

[0292] VDJ009 configuration:

[0293] Take a certain amount of VDJ009, dilute it with physiological saline for injection to a suitable concentration, and prepare it fresh for use.

[0294] Formulation of molding agent

[0295] Long-acting hIL-6:

[0296] Take a certain amount of hIL-6, dilute it to 12.5 μg / ml, and store it at -80℃.

[0297] Preparation of mBSA:

[0298] A certain amount of mBSA was accurately weighed using an electronic balance and dissolved in sterile water for injection to prepare a 70 mg / mL solution, which was prepared immediately before use.

[0299] 2 Experimental System

[0300] 2.1 Laboratory Animals

[0301] 2.2 Feeding and Management

[0302] Laboratory animals were housed in an SPF-grade animal barrier facility. Animals underwent at least 3 days of acclimatization before the experiment.

[0303] The ambient temperature and humidity of the barrier facility are controlled within the following ranges:

[0304] Temperature: 20-26℃

[0305] Humidity: 40-70%

[0306] Lighting: Alternates every 12 hours

[0307] Rat cages: Corn cob bedding is pressure-sterilized and cleaned, and changed weekly. Each cage has a label indicating the number of animals, sex, strain, reception time, group, and trial start time.

[0308] Feed and water: SPF grade growth and reproduction feed. Drinking water is sterile purified water. Animals have free access to sterile food and water.

[0309] Animal ID: Ear number

[0310] 3. Experimental Design

[0311] 3.1 Modeling and Grouping

[0312] Adaptation period: Before the start of the experiment, the test animals should undergo an adaptation period of at least 3 days at the test site.

[0313] Grouping: After the adaptation period, animals were randomly grouped according to their weight, with 5 animals per group, for a total of 6 groups, as shown in Table 22 below.

[0314] Table 22: Dosing regimens for animal experiments

[0315] Modeling procedure: After mice passed quarantine, on day 0, 10 μl of mBSA (70 mg / ml) was injected intra-articularly into the left knee joint cavity of the mice, while the control group received 10 μl of physiological saline. Subsequently, 20 μl of long-acting hIL-6 (250 ng) was injected subcutaneously into the left hind paw pad, while the control group received 20 μl of physiological saline. On day 3, long-acting hIL-6 was injected again, while the control group received physiological saline. On days 0 and 4, VDJ020, VDJ009, and physiological saline were injected into the tail vein of the mice. The diameter of the knee joint of the mice was measured using a digital caliper on days 4, 5, 6, and 7, and the mice were euthanized.

[0316] 3.2 Detection Indicators

[0317] 3.2.1 Clinical observation

[0318] Observe animals: All animals

[0319] Observation frequency and time: Observe twice a day during the experiment (with an interval of at least 6 hours between the two observations).

[0320] Observation content: including death and clinical manifestations.

[0321] 3.2.2 Weight

[0322] Animals tested: All animals

[0323] Detection time: before grouping, D0, D3, D6.

[0324] 3.2.3 Knee joint diameter

[0325] Animals tested: All animals

[0326] Testing time: The diameter of the left knee joint of the mouse was measured using a digital caliper on D4, D5, D6 and D7 respectively.

[0327] 4. Test Results

[0328] As shown in Figure 9, after administration on D0 and D4, VDJ009 at 1.14 mg / kg exhibited a slight inhibitory effect, while VDJ009 at 3.42 mg / kg and VDJ020 at 10 mg / kg showed similar inhibitory effects. VDJ009 at 10.26 mg / kg exerted a strong immunosuppressive effect, indicating that VDJ009 only requires one-third the dose of VDJ020 to achieve the same therapeutic effect. These results suggest that the antibody-drug conjugate of the present invention is beneficial for reducing dosage, improving patient tolerability, and reducing adverse reactions.

[0329] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody-drug conjugate having the structure represented by formula (I): Ab-(LD) n (I) in: Ab is an antibody against interleukin-6 receptor or its antigen-binding fragment, said antibody or its antigen-binding fragment comprising at least one light chain and at least one heavy chain, said light chain comprising at least one light chain variable region, said heavy chain comprising at least one heavy chain variable region; said heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 HCDR1, an amino acid sequence as shown in general formula (1) HCDR2, and an amino acid sequence as shown in SEQ ID NO:3 HCDR3; said light chain variable region having an amino acid sequence as shown in SEQ ID NO:4 LCDR1, an amino acid sequence as shown in SEQ ID NO:5 LCDR2, and an amino acid sequence as shown in SEQ ID NO:6 LCDR3; said general formula (1) has an amino acid sequence as shown in FISYSGX1TTYNPSLKS, wherein X1 is selected from G, V, F, K or R. D represents a drug. L represents the linking group used to connect Ab and D, and n is 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

2. The antibody-drug conjugate according to claim 1, characterized in that, The HCDR2 of the heavy chain variable region has an amino acid sequence as described in any one of SEQ ID NO:7, 9, 16, 19 and 20.

3. The antibody-drug conjugate according to claim 1, characterized in that, The heavy chain variable region includes an amino acid sequence as shown in any one of SEQ ID NO:25, 27, 34, 37 or 38 or an amino acid sequence having at least 85% sequence identity with it, and / or the light chain variable region has an amino acid sequence as shown in SEQ ID NO:60 or an amino acid sequence having at least 85% sequence identity with it.

4. The antibody-drug conjugate according to claim 1, characterized in that, The Fc region of the antibody has a mutation at positions 252, 254 and / or 256; Preferably, the Fc region of the antibody has mutations of M252Y, S254T and / or T256E; Preferably, the heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 62-66 or an amino acid sequence having at least 85% sequence identity with it, and / or, The light chain has an amino acid sequence as shown in SEQ ID NO:61 or an amino acid sequence having at least 85% sequence identity with it.

5. The antibody-drug conjugate according to claim 1, characterized in that, The antibody is a humanized antibody. Preferably, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. More preferably, the antibody is an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody.

6. The antibody-drug conjugate according to claim 1, characterized in that, The antibody or antigen-binding fragment has thermal stability and freeze-thaw stability.

7. The antibody-drug conjugate according to claim 1, characterized in that, The isoelectric point range of the antibody or antigen-binding fragment includes 9.0 to 10.

0.

8. The antibody-drug conjugate according to claim 1, characterized in that, D represents a glucocorticoid drug.

9. The antibody-drug conjugate according to claim 8, characterized in that, D has a part of the following formula (D1) or (D2): Where R1 is H, deuterium, or -H2PO3, and each occurrence of R is independently H, deuterium, or a halogen (e.g., F, Cl, Br). This indicates the site where D connects to L.

10. The antibody-drug conjugate according to claim 9, characterized in that, Equation (D2) is selected from the following equations (D2-a) to (D2-d):

11. The antibody-drug conjugate according to claim 1, characterized in that, The linker group (L) used to connect the antibody and the drug has a structure represented by the following formula (L): -C1-5 linear alkylene-(CO-NH-C1-5 linear alkylene) m -CO- formula (L) Where m is an integer from 1 to 5, for example, m is 1, 2, 3, 4 or 5; and optionally one or more methylene groups in the main chain of each of the C1-5 straight-chain alkylene groups are replaced by NH.

12. The antibody-drug conjugate according to claim 11, characterized in that, One or more hydrogens on the main chain of each of the C1-5 straight-chain alkylene groups are further reacted with R L Substituent substitution, where R L Selected from C1-5 alkyl groups, -(CH2) w -COOH and -(CH2) w -NH2, where w is an integer from 1 to 10, for example, w is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

13. The antibody-drug conjugate according to claim 11, characterized in that, The L is selected from any of the following structures:

14. The antibody-drug conjugate according to claim 11, characterized in that, The L is selected from any of the following structures:

15. The antibody-drug conjugate according to claim 1, characterized in that, The antibody-drug conjugate represented by formula (I) is represented by formula (Ia):

16. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1-15, and pharmaceutically acceptable excipients.

17. The pharmaceutical composition according to claim 16, characterized in that, The pharmaceutical composition comprises a drug / antibody ratio (DAR) of 1-10.

18. Use of the antibody-drug conjugate of any one of claims 1-15 or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating a disease selected from arthritis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa, chronic kidney disease anemia, atherosclerotic cardiovascular disease, thyroid eye disease, uveitis, and juvenile idiopathic arthritis.

19. A kit comprising: (a) an antibody-drug conjugate of any one of claims 1-15 or a pharmaceutical composition of claim 16 or 17; and (b) a specification indicating the use and usage information of the antibody-drug conjugate or pharmaceutical composition.

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