Protease-cleavable dual-lock antibody and use thereof

Abstract

Disclosed herein are a protease-cleavable dual-lock antibody and a use thereof, and in particular a protease-cleavable antibody, a nucleic acid comprising a nucleotide sequence encoding the protease-cleavable antibody, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or vector. Also disclosed are a pharmaceutical composition and conjugate comprising the protease-cleavable antibody, and a treatment method using the protease-cleavable antibody.

Description

Protease-cleavable double-locked antibody and its use

[0001] This international patent application claims priority to Chinese patent application No. 202410358603.2 filed on March 27, 2024, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0002] The present invention relates to protease-cleavable antibodies and uses of such antibodies, in particular their use in the treatment of cancer. Background Art

[0003] Antibodies are gamma globulins, primarily referred to as immunoglobulins (Ig). A monomeric antibody consists of two heavy chains and two light chains. The amino-termini of the polypeptide chains exhibit considerable variation in amino acid composition and are referred to as variable (V) domains / regions to distinguish them from the relatively constant (C) domains / regions. Each light chain has a variable domain (VL) and a constant domain (CL). Each heavy chain has four domains: a variable domain (VH) and constant domains 1 (CH1), 2 (CH2), and 3 (CH3). The antigen-binding site is located in the Fab (antigen-binding fragment) region, which includes the light chain variable domain (VL), the heavy chain variable domain (VH), the light chain constant domain (CL), and the heavy chain constant domain 1 (CH1). The combination of the light chain variable domain (VL) and the heavy chain variable domain (VH) is referred to as the Fv (variable fragment) region. The Fc (crystallizable fragment) region of an antibody includes heavy chain constant domains 2 and 3 (CH2 and CH3).

[0004] Each variable domain contains three highly variable loops, called complementarity determining regions (CDRs), evenly distributed between four less variable framework (FR) regions. It is the CDRs that provide the specific antigen recognition sites on the antibody surface, and the high variability of these regions enables antibodies to recognize an almost unlimited number of antigens. The heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetrical structure. The hinge region is the heavy chain region between the first and second constant domains (CH1 and CH2) and is held together by disulfide bonds. This flexible hinge region allows the distance between the two antigen binding sites to change.

[0005] In various clinical settings such as cancer therapy, it is often desirable to selectively destroy a single target cell or a specific target cell type. One way to achieve this is by inducing an immune response against the tumor, for example, by administering antibody molecules to cause immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL) to attack and destroy tumor cells. However, escaping anti-tumor immunity is a hallmark of tumors, and thus various types of tumor immunotherapies have been developed, such as immune checkpoint inhibitors (ICIs) and T cell redirecting bispecific antibodies (TCBS). Although immunotherapy has prolonged the survival of patients with various types of tumors, the expression of some antigens in healthy tissues causes systemic immune activation effects to also occur in healthy tissues, resulting in on-target toxicity and immune-related adverse events (irAES).

[0006] Therefore, there is a need for new antibody formats with improved safety profiles and increased safety windows. Summary of the Invention

[0007] The present invention provides a novel protease-cleavable double-lock antibody, in which each Fab is linked to a masking peptide via a different protease-cleavable site, thereby masking the antibody's antigen-binding site. These protease-cleavable sites are expressed or highly expressed in tumor microenvironments or inflammatory environments, while not expressed or expressed at low levels in normal tissues. This allows the masking peptide to be fully cleaved in tumor tissue or at the site of inflammation, releasing the antigen-binding site, allowing it to bind to the target antigen and exert its function.

[0008] The Double-Lock antibody format of the present invention, comprising two distinct protease cleavage sites, can reduce on-target toxicity and improve safety. Double-Lock antibodies require the release of two locks (two distinct protease cleavage sites, such as MMP and uPA cleavage sites) to fully release the antibody's specific activity. The rate of fully cleaved antibodies in normal tissues is low, thus providing a higher safety window.

[0009] The double-lock antibody format has a wider range of applications in cancer patients with liver damage, inflammation, etc. For example, because MMP is highly expressed in areas of liver damage, inflammation, etc., antibodies containing only one protease cleavage site will release their activity non-specifically in the liver of cancer patients with liver disease or inflammation, causing off-target toxicity. However, after being cleaved by MMP in the liver, the double-lock antibody format only exposes the antibody activity of one Fab. For most antagonistic antibodies, their monovalent activity is limited. For agonistic antibodies (such as TREM2, TREM1 and CD40 agonistic antibodies that have entered the clinic), their activity requires a multivalent form of the antibody. Monovalent agonistic antibodies are inactive. Therefore, the double-lock antibody format can significantly reduce off-target toxicity and improve safety.

[0010] In addition, the CrossMAb technology is used to interchange the HC and LC domains to solve the LC / HC mismatch problem while retaining the original antigen affinity; and the knob-into-hole structure is introduced into the antibody to promote the heterologous dimerization of the two heavy chains.

[0011] Accordingly, in one aspect, the present invention provides a protease-cleavable antibody comprising:

[0012] a first Fab comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and

[0013] a second Fab comprising a second heavy chain variable region (VH2) and a second light chain variable region (VL2);

[0014] wherein said VH1 and / or said VL1 are linked to a masking peptide via a first linker comprising a first protease cleavage site;

[0015] wherein said VH2 and / or said VL2 are linked to the masking peptide via a second linker comprising a second protease cleavage site;

[0016] The first protease cleavage site is specifically cleaved by a first protease, the second protease cleavage site is specifically cleaved by a second protease, and the first protease and the second protease are different.

[0017] In some embodiments of the antibodies disclosed herein, the VH1 and / or the VL1 are linked at their N-termini to the masking peptide via the first linker, and the VH2 and / or the VL2 are linked at their N-termini to the masking peptide via the second linker.

[0018] In some embodiments, the VH1 and the VL1 are linked at their N-termini to the masking peptide via the first linker, and the VH2 and the VL2 are linked at their N-termini to the masking peptide via the second linker.

[0019] In some embodiments, the first protease and the second protease are each independently a protease expressed or overexpressed in a tumor microenvironment or an inflammatory environment.

[0020] In some embodiments, the first protease and the second protease are each independently selected from the group consisting of a serine protease, a metalloprotease, a thiol protease, and a carboxyl protease.

[0021] In some embodiments, the first protease and the second protease are each independently selected from uPA, MMP, TEV protease, plasmin, thrombin, FXa, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM, ADAMTS, caspase, enterokinase, and HRV 3C protease.

[0022] In some embodiments, one of the first protease and the second protease is a serine protease and the other is a metalloprotease.

[0023] In some embodiments, one of the first protease and the second protease is uPA and the other is a MMP (eg, selected from MMP2 and MMP9).

[0024] In some embodiments, the first protease cleavage site and the second protease cleavage site are each independently selected from SEQ ID NOs: 23-32, preferably selected from SEQ ID NO: 23 and SEQ ID NO: 24.

[0025] In some embodiments, the first linker and the second linker each independently comprise an amino acid sequence selected from SEQ ID NOs: 23-42.

[0026] In some embodiments, the masking peptide is capable of inhibiting binding of the antibody to its targeted antigen and has a length of 5-60 amino acids.

[0027] In some embodiments, the masking peptide inhibits the binding of the antibody to its targeted antigen by steric hindrance and / or binding to the antibody's antigen binding site.

[0028] In some embodiments, the masking peptide is selected from the group consisting of a coiled coil-forming peptide, an antibody hinge region, an antibody fragment (e.g., dsFv), a non-antibody protein fragment (e.g., the LAP domain from TGF-β), an affinity polypeptide capable of binding to the antigen binding site of the antibody, and a polypeptide comprising an antigenic epitope to which the antibody binds, preferably selected from the group consisting of a coiled coil-forming peptide and an antibody hinge region.

[0029] In some embodiments, the coiled-coil-forming peptide is a heterodimeric coiled-coil-forming peptide (e.g., a sequence pair selected from the group consisting of SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54) or a homodimeric coiled-coil-forming peptide (e.g., SEQ ID NO: 77).

[0030] In some embodiments, the antibody hinge region is a human antibody hinge region, eg, comprising the amino acid sequence of SEQ ID NO: 78.

[0031] In some embodiments, the VH and VL in the first Fab or the second Fab are replaced with each other. In some embodiments, the CH1 and CL in the first Fab or the second Fab are replaced with each other. In some embodiments, the VH and VL in the first Fab or the second Fab are replaced with each other and the CH1 and CL are replaced with each other.

[0032] In some embodiments, the antibody comprises:

[0033] A first heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a first linker, VH1, CH1, CH2, and CH3; and a first light chain comprising, from N-terminus to C-terminus, a masking peptide, a first linker, VL1, and CL; and a second heavy chain and a second light chain selected from:

[0034] (i) a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, CL, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, and CH1;

[0035] (ii) a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, CH1, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, and CL; or

[0036] (iii) a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, CL, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, and CH1.

[0037] In some embodiments, one or both of the CH3s contain at least one amino acid mutation capable of promoting heterodimerization of the first heavy chain and the second heavy chain; preferably, one of the CH3s contains T366S, L368A and Y407V mutations, and the other contains T366W mutation.

[0038] In some embodiments, the CH1 and CL comprise amino acid mutations capable of promoting dimerization of the heavy and light chains; preferably, the CH1 comprises K158E and K224E mutations, and the CL comprises E143R and Q144K mutations (according to IMTG numbering).

[0039] In some embodiments, the first Fab and the second Fab bind to the same antigen.In some embodiments, the antigen is selected from a tumor associated antigen (TAA) and a proinflammatory cytokine.

[0040] In some embodiments, the first Fab and the second Fab bind to different antigens, and the first Fab and the second Fab have KD values ​​that are at least 10 -8 The binding affinity of M. In some embodiments, one of the antigens is selected from the group consisting of TAAs and proinflammatory cytokines, and the other is selected from the group consisting of TAAs, proinflammatory cytokines, and immune cell antigens (e.g., T cell antigens).

[0041] In some embodiments, the TAA is CD20, and wherein the VH1 and VH2 comprise HCDRs 1-3 having the following amino acid sequences, respectively: GYTFTSYN (SEQ ID NO: 17), IYPGNGDT (SEQ ID NO: 18), and AR, and the VL1 and VL2 comprise LCDRs 1-3 having the following amino acid sequences, respectively: SSVSY (SEQ ID NO: 20), ATS, and QQWTSNP (SEQ ID NO: 21).

[0042] In some embodiments, the VH1 and VH2 comprise the amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19, and the VL1 and VL2 comprise the amino acid sequence as set forth in SEQ ID NO: 22, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22.

[0043] In some embodiments, the antibody comprises:

[0044] (i) a first heavy chain comprising amino acids as set forth in SEQ ID NO:5, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:5; a second heavy chain comprising amino acids as set forth in SEQ ID NO:6, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:6; a first light chain comprising amino acids as set forth in SEQ ID NO:7, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:7; and a second light chain comprising amino acids as set forth in SEQ ID NO:8, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:8; or

[0045] (ii) a first heavy chain comprising amino acids as set forth in SEQ ID NO:9, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:9; a second heavy chain comprising amino acids as set forth in SEQ ID NO:10, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:10; a first light chain comprising amino acids as set forth in SEQ ID NO:11, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:11; and a second light chain comprising amino acids as set forth in SEQ ID NO:12, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:12; or

[0046] (iii) a first heavy chain comprising amino acids as set forth in SEQ ID NO: 13, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 13; a second heavy chain comprising amino acids as set forth in SEQ ID NO: 14, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 14; a first light chain comprising amino acids as set forth in SEQ ID NO: 15, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 15; and a second light chain comprising amino acids as set forth in SEQ ID NO: 16, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 16.

[0047] In another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody disclosed herein.

[0048] In yet another aspect, the present invention provides a vector comprising a nucleic acid disclosed herein.

[0049] In yet another aspect, the invention provides a host cell comprising a nucleic acid or a vector disclosed herein.

[0050] In yet another aspect, the present invention provides a pharmaceutical composition comprising (i) an antibody disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0051] In some embodiments of the pharmaceutical composition disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent.

[0052] In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0053] In yet another aspect, the present invention provides a conjugate comprising an antibody disclosed herein and a chemical moiety conjugated thereto.

[0054] In some embodiments of the presently disclosed conjugates, the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

[0055] In yet another aspect, the present invention provides a method of treating a disease in a subject, comprising administering to the subject an effective amount of an antibody disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein.

[0056] In some embodiments of the methods disclosed herein, the disease is cancer or an inflammatory disease.

[0057] In some embodiments, the cancer is selected from cervical cancer, lung cancer, liver cancer, breast cancer, and colon cancer.

[0058] In some embodiments, the inflammatory disease is selected from rheumatoid arthritis, drug-induced hepatitis, liver fibrosis, chronic obstructive pulmonary disease, asthma, and atopic dermatitis.

[0059] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.

[0060] In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] An understanding of the features and advantages of the present invention may be obtained by reference to the following detailed description which illustrates exemplary embodiments utilizing the principles of the invention and the accompanying drawings, in which:

[0062] FIG1 shows a schematic structural diagram of molecule 1.

[0063] FIG2 shows a schematic diagram of the structure of molecule 2.

[0064] FIG3 shows a schematic diagram of the structure of molecule 3.

[0065] FIG4 shows a schematic diagram of the structure of molecule 4.

[0066] FIG5 shows the SDS-PAGE results of molecule 1.

[0067] FIG6 shows the SEC-HPLC results of molecule 1.

[0068] FIG7 shows the SDS-PAGE results of molecule 2.

[0069] FIG8 shows the SEC-HPLC results of molecule 2.

[0070] FIG9 shows the SDS-PAGE results of molecule 3.

[0071] FIG10 shows the SEC-HPLC results of molecule 3.

[0072] FIG11 shows the SDS-PAGE results of molecule 4.

[0073] FIG12 shows the SEC-HPLC results of molecule 4.

[0074] Figure 13 shows the binding of molecule 2 to Raji cells before and after enzyme cleavage as determined by FACS. Molecule 1 was used as a positive control.

[0075] Figure 14 shows the binding of molecule 3 to Raji cells before and after enzyme cleavage as determined by FACS. Molecule 1 was used as a positive control.

[0076] Figure 15 shows the binding of molecule 4 to Raji cells before and after enzyme cleavage as determined by FACS. Molecule 1 was used as a positive control.

[0077] FIG16 shows the ADCC activity of molecule 4 on Raji cells before and after enzyme cleavage as determined by the EuTDA method. DETAILED DESCRIPTION

[0078] The above features and advantages of the present invention and additional features and advantages will be more clearly understood from the following detailed description of embodiments taken in conjunction with the accompanying drawings.

[0079] The embodiments described herein with reference to the accompanying drawings are illustrative, exemplary, and are used for a general understanding of the present invention. The embodiments should not be construed as limiting the scope of the present invention. Identical or similar elements and elements with identical or similar functions are represented by the same reference numerals throughout the specification.

[0080] Unless otherwise mentioned or defined, all terms used have the ordinary meaning in the art that is clear to those skilled in the art. Reference is made, for example, to standard manuals, such as Leuenberger, HGW, Nagel, B. and Klbl, H. eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., "Current protocols in molecular biology", Green Publishing and Wiley InterScience, New York (1987); Roitt et al., "Immunology (6th Ed.), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York (2005), and the general background art cited above.

[0081] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies and in some embodiments, reference to "an antibody" includes a plurality of antibodies, and so forth.

[0082] Unless otherwise stated or defined, the term “comprise” and variations such as “include” and “comprising” will be understood to imply the inclusion of stated elements or steps or groups of elements or steps but not the exclusion of any other elements or steps or groups of elements or steps.

[0083] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Such molecules typically comprise two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (or domain) (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (or domain) (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains of an antibody contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q (the first component in the classical pathway of complement activation).

[0084] The heavy chain of an immunoglobulin can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment). The Fd region comprises the VH and CH1 domains and combines with the light chain to form the Fab (antigen binding fragment). The Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to the cognate Fc receptors of effector cells. The hinge region found in IgG, IgA, and IgD immunoglobulin classes acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region. The hinge domain is structurally diverse, with different sequences and lengths between immunoglobulin classes and subclasses.

[0085] A "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of a "framework" region separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs that specifically bind to an antigenic epitope. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0086] The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 878-883, 1989); a composite of the Chothia Kabat CDR, wherein CDR-H1 is a composite of the Chothia CDR and the Kabat CDR; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (Kabat numbering system), in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure may utilize CDRs defined according to any of these numbering systems, but preferred embodiments utilize the Kabat defined CDRs.

[0087] As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit the desired biological activity.

[0088] The term "bispecific antibody" in the context of the present invention is understood to mean an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a target.

[0089] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Avidity represents the equilibrium constant (KD) for the dissociation of an antigen from an antibody and is a measure of the binding strength between an antigenic determinant and the antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD.

[0090] Avidity is a measure of the strength of binding between an antibody and its associated antigen. Avidity is related to the affinity between an antigenic determinant and the antigen-binding site of an antibody and the number of associated binding sites present on the antibody. Typically, an antibody will bind to an antigen with the following dissociation constant (KD): 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10 -8 M to 10 -12 M, and / or have the following binding affinity: at least 10 7 M -1 , preferably at least 10 8 M - 1 , more preferably at least 10 9 M -1 , such as at least 10 12 M -1 It is generally believed that any value greater than 10 -4 M's K D The values ​​represent non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including for example Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assays, and different variations thereof known in the art.

[0091] The term "epitope" refers to the site on an antigen to which an antibody binds. An epitope can be formed by continuous amino acids or by non-continuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by continuous amino acids (also referred to as linear epitopes) are typically retained in exposure to denaturing solvents, while epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost in the treatment of denaturing solvents. An epitope typically includes at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. An epitope defines the minimum binding site of an antibody and is therefore the specific target of an antibody or its antigen-binding fragment.

[0092] As used herein, the term "protease," also known as endopeptidase, acts on peptide bonds within peptide chains, generating shorter peptide chains containing fewer amino acids. Proteases are specific for the peptide bonds formed with different amino acids. Proteases can be divided into four categories based on their catalytic mechanism, as shown in the table below.

[0093] As used herein, the term "a protease expressed or overexpressed in a tumor microenvironment or an inflammatory environment" is a protease that is differentially expressed in a tumor microenvironment or an inflammatory environment compared to normal tissue.

[0094] Urokinase-type plasminogen activator (uPA) is a serine protease involved in tissue remodeling and cell migration. It is highly expressed in different tumor microenvironments and is a biomarker associated with cancer prognosis. Matrix metalloproteinases (MMPs) play a key role in metastasis and are associated with survival in various cancers. They are highly expressed in different tumor microenvironments, as well as in sites of liver injury and inflammation.

[0095] As used herein, the term "coiled-coil-forming peptide" refers to a pair of peptides that can bind to each other to form a coiled-coil. "Coiled-coil" is a term used in the art to refer to a bundle of α-helices that are wound into a superhelical structure. In this context, the coiled-coil formed is typically formed by two coiled-coil-forming peptides. Coiled-coils are formed by α-helices on the peptides in parallel or opposite directions.

[0096] As used herein, the term "tumor-associated antigen" or "TAA" refers to an antigen that is differentially expressed in cancer cells compared to normal cells and, therefore, can be used to target cancer cells.

[0097] As used herein, the term "proinflammatory cytokines" refers to a class of cytokines that can promote inflammation. Common proinflammatory cytokines include IL-1, IL-2, IL-6, IL-8, IL-12, TNF-α, IFN-γ, and macrophage migration inhibitory factor (MIF).

[0098] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acids) have identical residues at identical positions when aligned. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the % identity of the amino acid sequence to SEQ ID NO: Y, and is stated as X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y.

[0099] Computer programs are typically used for such calculations. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0100] Furthermore, when determining the degree of sequence identity between two amino acid sequences, one of skill may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, which has little or essentially no effect on the function, activity or other biological properties of the polypeptide.

[0101] Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0102] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0103] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0104] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0105] The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and not largely toxic to the recipient thereof, and / or such carrier or adjuvant is approved or available for inclusion in pharmaceutical compositions for parenteral administration to humans.

[0106] As used herein, the terms "treat," "therapy," "treatment," and the like refer to the administration of an agent or the performance of a procedure for the purpose of obtaining an effect. These effects may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of achieving a partial or complete cure of a disease and / or disease symptoms. As used herein, "treatment" may include treating a disease or condition (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease or disease symptom in a subject who may be susceptible to the disease (e.g., including a disease that may be associated with or caused by the primary disease) but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any indicator of success in the treatment, improvement, or prevention of cancer, including any objective or subjective parameter, such as a reduction in symptoms; relief; elimination of disease symptoms or making the disease condition more tolerable for the patient; slowing the rate of deterioration or decline; or reducing the final stage of deterioration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes the administration of an antibody, composition, or conjugate disclosed herein to prevent or delay, alleviate, or arrest or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptom, or disease side effect in a subject.

[0107] As used herein, the term "effective amount" refers to the amount administered to a subject for treating a disease that is sufficient to effect treatment for the disease.

[0108] As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock, and laboratory, zoo, sports, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

[0109] The Double-lock antibody format of the present invention can reduce on-target toxicity and off-target toxicity by containing two different protease cleavable sites in two Fabs, thereby improving safety and medication safety window.

[0110] Accordingly, in one aspect, the present invention provides a protease-cleavable antibody comprising:

[0111] a first Fab comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and

[0112] a second Fab comprising a second heavy chain variable region (VH2) and a second light chain variable region (VL2);

[0113] wherein said VH1 and / or said VL1 are linked to a masking peptide via a first linker comprising a first protease cleavage site;

[0114] wherein said VH2 and / or said VL2 are linked to the masking peptide via a second linker comprising a second protease cleavage site;

[0115] The first protease cleavage site is specifically cleaved by a first protease, the second protease cleavage site is specifically cleaved by a second protease, and the first protease and the second protease are different.

[0116] In some embodiments of the antibodies disclosed herein, the VH1 and / or the VL1 are linked at their N-termini to the masking peptide via the first linker, and the VH2 and / or the VL2 are linked at their N-termini to the masking peptide via the second linker.

[0117] In some embodiments, the VH1 and the VL1 are linked at their N-termini to the masking peptide via the first linker, and the VH2 and the VL2 are linked at their N-termini to the masking peptide via the second linker.

[0118] In some embodiments, the VH1 or the VL1 is linked at its N-terminus to the masking peptide via the first linker, and the VH2 or the VL2 is linked at its N-terminus to the masking peptide via the second linker.

[0119] In some embodiments, the first protease and the second protease are each independently a protease expressed or overexpressed in a tumor microenvironment or an inflammatory environment.

[0120] Protease can be any suitable protease known in the art that is expressed or overexpressed in a tumor microenvironment or inflammatory environment. In some embodiments, the first protease and the second protease are each independently a protease that is expressed or overexpressed in a tumor microenvironment. In some embodiments, the first protease and the second protease are each independently a protease that is expressed or overexpressed in an inflammatory environment.

[0121] In some embodiments, the first protease and the second protease are each independently selected from a serine protease, a metalloprotease, a thiol protease and a carboxy protease. In some embodiments, the first protease and the second protease are each independently selected from a serine protease and a metalloprotease. In some embodiments, one of the first protease and the second protease is a serine protease and the other is a metalloprotease.

[0122] Examples of serine proteases include, but are not limited to, urokinase-type plasminogen activator (uPA), thrombin, activated protein C, cathepsin A, cathepsin G, chymase, and coagulation factors (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa).

[0123] Examples of metalloproteinases include, but are not limited to, matrix metalloproteinases (MMPs) (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and MMP-27), methyldopa (Meprin), neprilysin, PSMA, and BMP-1.

[0124] Thiol proteases are also known as cysteine ​​proteases, examples of which include, but are not limited to, Cruzipain, Legumin, Otubain-2, Cathepsin B, Cathepsin H, Cathepsin L.

[0125] Carboxyl proteases are also called acidic proteases, and examples thereof include, but are not limited to, aspartic proteases (eg, RACE, renin, pepsin, cathepsin D, and cathepsin E), and glutamic proteases.

[0126] In some embodiments, the first protease and the second protease are each independently selected from urokinase-type plasminogen activator (uPA); matrix metalloproteinases (MMPs) (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and MMP-27); tobacco etch virus (TEV) protease; plasmin; thrombin; prostate-specific antigen (PSA) enzyme; PSMA; ADAM / ADAMTS (e.g., ADAM 8, ADAM 9, ADAM10, ADAM12, ADAMIS, ADAM17 / TACE, ADAMDECI, ADAMTS1, ADAMTS4, and ADAMTS5); caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase- cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and cathepsin X / Z / P); cysteine ​​proteases (e.g., cruzipain, legumin, and otubain-2); KLK (e.g., such as KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14); metalloproteinases (such as methyldopa (Meprin), enkephalinase, PSMA, and BMP-1); serine proteases (such as activated protein C, cathepsin A, cathepsin G, chymase, and coagulation factors (such as FVIIa, FIXa, FXa, FXIa, FXIIa)); elastase; granules enzyme B; guanidinobenzoate esterase; HtrA1; human neutrophil elastase; lactoferrin; marapsin; hepatitis C virus (HCV)-NS3 / 4 serine protease; PACE4; tPA; tryptase; type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, Hepsin, intercalated proteinase-2, MT-SP1 / intercalated proteinase, TMPRSS2, TMPRSS3, and TMPRSS4);Matriptase; Asparagine endopeptidase legumain; Enterokinase; Human rhinovirus (HRV) 3C protease (e.g., PreScission).

[0127] In some embodiments, the first protease and the second protease are each independently selected from urokinase-type plasminogen activator (uPA), MMP (e.g., MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14), TEV protease, plasmin, thrombin, FXa, PSA, PSMA, cathepsins (cathepsin D, cathepsin K, cathepsin S), ADAM / ADAMT ( ADAM10, ADAM12, ADAMTS, TACE), caspases (caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14), enterokinase, and HRV 3C protease.

[0128] In some embodiments, the first protease and the second protease are each independently selected from urokinase-type plasminogen activator (uPA), MMP (e.g., MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14), TEV protease, thrombin, FXa, enterokinase, and HRV 3C protease.

[0129] In some embodiments, one of the first and second proteases is uPA and the other is a MMP. In some embodiments, the MMP is selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and MMP-27. In some embodiments, the MMP is selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-14. In some embodiments, the MMP is selected from the group consisting of MMP-2 and MMP-9. In some embodiments, one of the first and second proteases is uPA and the other is MMP2. In some embodiments, one of the first and second proteases is uPA and the other is MMP9. In some embodiments, one of the first protease and the second protease is uPA, and the other is MMP2 and MMP9. In some embodiments, one of the first protease and the second protease is uPA, and the other is TEV protease. In some embodiments, one of the first protease and the second protease is a MMP, and the other is TEV protease.

[0130] The protease cleavage site is any suitable cleavage site that can be specifically cleaved by the proteases described herein. The first protease cleavage site is a cleavage site that is specifically cleaved by the first protease described herein. The second protease cleavage site is a cleavage site that is specifically cleaved by the second protease described herein.

[0131] In some embodiments, the first protease cleavage site and the second protease cleavage site are each independently selected from the protease cleavage site by the following protease specific cutting: serine protease, metalloprotease, thiol protease and carboxyl protease. In some embodiments, the first protease cleavage site and the second protease cleavage site are each independently selected from the protease cleavage site by serine protease and metalloprotease specific cutting. In some embodiments, one of the first protease cleavage site and the second protease cleavage site is a protease cleavage site by serine protease specific cutting, and another is a protease cleavage site by metalloprotease specific cutting.

[0132] In some embodiments, the first protease cleavage site and the second protease cleavage site are each independently selected from protease cleavage sites specifically cleaved by: urokinase-type plasminogen activator (uPA); matrix metalloproteinases (MMPs) (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and MMP-27); tobacco etch virus (TEV) protease; plasmin; thrombin; prostate-specific antigen (PSA) enzyme; PSMA; ADAM / ADAMTS (e.g., ADAM 8, ADAM 9, ADAM10, ADAM12, ADAMIS, ADAM17 / TACE, ADAMDECI, ADAMTS1, ADAMTS4, and ADAMTS5); caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10); , caspase-11, caspase-12, caspase-13, and caspase-14); aspartic proteases (e.g., RACE and renin); aspartic cathepsins (e.g., cathepsin D and cathepsin E); cysteine ​​cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and cathepsin X / Z / P); cysteine Proteases (e.g., Cruzipain, Leguminosin, and Otubain-2); KLKs (e.g., KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14); Metalloproteinases (e.g., Meprin, Neprilysin, PSMA, and BMP-1); Serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase, and coagulation factors (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa)); Elastase; Granzyme B; Guanidinobenzoate esterase; HtrA1; human neutrophil elastase; Lactoferrin; Marapsin; Hepatitis C virus (HCV)-NS3 / 4 serine protease; PACE4; tPA; Tryptase;Type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, Hepsin, Interstitial proteinase-2, MT-SP1 / Interstitial proteinase, TMPRSS2, TMPRSS3, and TMPRSS4); matriptase; asparagine endopeptidase legumain; enterokinase; human rhinovirus (HRV) 3C protease (e.g., PreScission).

[0133] In some embodiments, the first protease cleavage site and the second protease cleavage site are each independently selected from the group consisting of protease cleavage sites specifically cleaved by urokinase-type plasminogen activator (uPA), MMPs (e.g., MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14), TEV protease, plasmin, thrombin, FXa, PSA, PSMA, cathepsins (cathepsin D, cathepsin K, cathepsin B), and protease inhibitors. S), ADAM / ADAMT (ADAM10, ADAM12, ADAMTS, TACE), caspases (caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14), enterokinase, and HRV 3C protease.

[0134] In some embodiments, the first protease cleavage site and the second protease cleavage site are each independently selected from protease cleavage sites specifically cleaved by the following proteases: urokinase-type plasminogen activator (uPA), MMP (e.g., MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14), TEV protease, thrombin, FXa, enterokinase, and HRV 3C protease.

[0135] In some embodiments, one of the first protease cleavage site and the second protease cleavage site is a cleavage site specifically cleaved by uPA, and the other is a cleavage site specifically cleaved by MMP. In some embodiments, one of the first protease cleavage site and the second protease cleavage site is a cleavage site specifically cleaved by uPA, and the other is a cleavage site specifically cleaved by MMP2 / MMP9. In some embodiments, one of the first protease cleavage site and the second protease cleavage site is a cleavage site specifically cleaved by uPA, and the other is a cleavage site specifically cleaved by TEV protease. In some embodiments, one of the first protease cleavage site and the second protease cleavage site is a cleavage site specifically cleaved by MMP, and the other is a cleavage site specifically cleaved by TEV protease.

[0136] The cleavage sites of the proteases described herein are known in the art. Commonly used exemplary protease cleavage sites are shown in the table below.

[0137] In some embodiments, the first protease cleavage site and the second protease cleavage site are each independently selected from SEQ ID NOs: 23 to 32. In a preferred embodiment, one of the first protease cleavage site and the second protease cleavage site is SEQ ID NO: 23, and the other is SEQ ID NO: 24.

[0138] In some embodiments, the first linker comprises or consists of a first protease cleavage site. In some embodiments, the second linker comprises or consists of a second protease cleavage site.

[0139] In some embodiments, the first linker comprises: L-CS1, wherein L represents an optional linker and CS1 represents a first protease cleavage site. In some embodiments, the second linker comprises: L-CS2, wherein L represents an optional linker and CS2 represents a second protease cleavage site. The linker can be any suitable linker known in the art, such as a flexible linker. In some embodiments, the linker is (GGGS)n or (GGGGS)n, wherein n is an integer selected from 1-4. In some embodiments, the linker is GGGS (SEQ ID NO: 43). In a preferred embodiment, the linker is GGGGS (SEQ ID NO: 44).

[0140] In some embodiments, the first linker and the second linker each independently comprise an amino acid sequence selected from the group consisting of: SGRSA (SEQ ID NO:23), PLGLAG (SEQ ID NO:24), IPVSLRSG (SEQ ID NO:25), GPLGVR (SEQ ID NO:26), ENLYFQG (SEQ ID NO:27), LVPRGS (SEQ ID NO:28), IEGR (SEQ ID NO:29), IDGR (SEQ ID NO:30), DDDDK (SEQ ID NO:31), LEVLFQGP (SEQ ID NO:32), GGGGSSGRSA (SEQ ID NO:33), GGGGSPLGLAG (SEQ ID NO:34), GGGGSIPVSLRSG (SEQ ID NO:35), GGGGSGPLGVR (SEQ ID NO:36), GGGGSENLYFQG (SEQ ID NO:37), GGGGSLVPRGS (SEQ ID NO:38), GGGGSIEGR (SEQ ID NO:39), GGGGSIDGR (SEQ ID NO:40). ID NO:40), GGGGSDDDDK (SEQ ID NO:41), GGGGSLEVLFQGP (SEQ ID NO:42).

[0141] In a preferred embodiment, the first linker and the second linker each independently comprise an amino acid sequence selected from SEQ ID NOs: 23-24 and 33-34.

[0142] In this article, the masking peptide can be any polypeptide known in the art that can inhibit the binding between an antibody and its targeted antigen. The connection of the masking peptide to the antibody can reduce the binding affinity of the antibody by, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 1500, 2000, 4000, 5000 or 10000 times (relative to the same antibody without such connection or after such connection is cut). Before being cleaved by the protease, the masking peptide can inhibit the binding between the antibody and its targeted antigen by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to the antibody of the present invention after being specifically cleaved by the protease, and when the antibody is cleaved by the protease, the peptide mask cannot inhibit the binding between the antibody and its targeted antigen.

[0143] In some embodiments, the masking peptide is capable of inhibiting the binding between the antibody and its targeted antigen. In some embodiments, the masking peptide is capable of inhibiting the binding between a first Fab and its antigen and is capable of inhibiting the binding between a second Fab and its antigen. In some embodiments, the masking peptide that inhibits the binding between the first Fab and its antigen and the masking peptide that inhibits the binding between the second Fab and its antigen may be the same or different.

[0144] In some embodiments, the masking peptide has a length of 5-60 amino acids. For example, the masking peptide has a length of 5-55, 5-50, 5-45, 5-40, 8-60, 8-55, 8-50, 8-45, 8-40, 10-60, 10-55, 10-50, 10-45, 10-40, 12-60, 12-55, 12-50, 12-45, 12-40, 15-60, 15-55, 15-50, 15-45, or 15-40 amino acids. In some embodiments, the masking peptide has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.

[0145] In some embodiments, the masking peptide inhibits the binding of the antibody to its targeted antigen by steric hindrance and / or binding to the antibody's antigen binding site.

[0146] In some embodiments, the masking peptide inhibits the binding of the antibody to its targeted antigen by steric hindrance. Examples of such masking peptides include, but are not limited to, coiled-coil forming peptides, antibody hinge regions, antibody fragments, and non-antibody protein fragments.

[0147] The coiled-coil-forming peptides are a pair of peptides that can bind to each other to form a coiled-coil, and sterically block the binding of antibodies to antigens by forming a coiled-coil secondary structure.

[0148] In the present invention, the coiled coil formed is generally formed by two coiled coil forming peptides. Leucine zipper forming peptides are an example of peptides that combine to form coiled coils. Coiled coils are formed by alpha helices on the peptide in parallel or opposite directions. A further feature of the coiled coil is that the core of the fiber bundle is filled with amino acid side chains, called "knobs-into-holes", in which the residues from one helix (knobs) are filled into the space (hole) surrounded by four side chains facing the helix. The residues involved in the knob-into-hole interaction are generally hydrophobic, while the outer residues are hydrophilic, so the chemical properties of the side chains of the coiled coil sequence show "heptad" repeats. WO2011034605 provides an example of a consensus formulae for heptad repeats in coiled coil forming peptides.

[0149] Formula 1: (X1, X2, X3, X4, X5, X6, X7)n

[0150] X1 is a hydrophobic amino acid or asparagine;

[0151] X2, X3, and X6 are any amino acids;

[0152] X4 is a hydrophobic amino acid;

[0153] X5 and X7 are each a charged amino acid residue.

[0154] Formula 2: (X1', X2', X3', X4', X5', X6', X7')n

[0155] X1′ is a hydrophobic amino acid or asparagine;

[0156] X2', X3' and X'6 are each any amino acid residue;

[0157] X4′ is a hydrophobic amino acid;

[0158] X5' and X7' are each charged amino acid residues;

[0159] wherein n in Formulas 1 and 2 is greater than or equal to 2; and wherein each heptad repeat in the first coiled-coil-forming peptide comprises an X5 residue having an opposite charge to the X7' residue in the second coiled-coil-forming peptide, and each heptad repeat in the first coiled-coil-forming peptide comprises an X7 residue having an opposite charge to the X5' residue in the second coiled-coil-forming peptide. The heptad repeats in the coiled-coil-forming peptides may be identical or different and conform to Formula 1 or 2.

[0160] Coiled coils can be homodimers or heterodimers. Heterodimeric coiled coil forming peptides can include parallel heterodimers and antiparallel heterodimers. Homodimeric coiled coil forming peptides can be helix-turn-helix homodimers.

[0161] The heterodimeric coiled-coil forming peptide can be, for example, a sequence pair selected from the group consisting of VDELQAEVDQLEDENYALKTKVAQLRKKVEKL (SEQ ID NO:45) and VAQLEEKVKTLRAENYELKSEVQRLEEQVAQL (SEQ ID NO:46), GKIAALKQKIAALKYKNAALKKKIAALKQ (SEQ ID NO:47) and GEIAALEQEIAALEKENAALEWEIAALEQ (SEQ ID NO:48), EIAALEKENAALEWEIAALEQ (SEQ ID NO:49) and KIAALKYKNAALKKKIAALKQ (SEQ ID NO:50), EACGASTSVDELQAEVDQLEDENYALKTKVAQLRKKVEKL (SEQ ID NO:51) and EACGASTTVAQLEEKVKTLRAENYELKSEVQRLEEQVAQL (SEQ ID NO:52). NO:52), AQLKKKLQANKKELAQLKWKLQALKKKLAQ (SEQ ID NO:53) and AQLEKELQALEKKLAQLEWENQALEKELAQ (SEQ ID NO:54).

[0162] The heterodimeric coiled coil-forming peptides can also be sequence pairs selected from the following: GASTSVDELQAEVDQLQDENYALKTKVAQLRKKVEKLSE (SEQ ID NO:55) and GASTTVAQLRERVKTLRAQNYELESEVQRLREQVAQLA (SEQ ID NO:56); EACGASTSVDELQAEVDQLQDENYALKTKVAQLRKKVEKLSE (SEQ ID NO:57) and EACGASTTVAQLRERVKTLRAQNYELESEVQRLREQVAQLA (SEQ ID NO:58); LEIEAAFLERENTALETRVAELRQRVQRARNRVSQYRTRY (SEQ ID NO:59) and LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRY (SEQ ID NO:60); EACGALEIEAAFLERENTALETRVAELRQRVQRARNRVSQYRTRY (SEQ ID NO:61) and EACGALEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRY (SEQ ID NO:62); LEIRAAFLRRRNTALRTRVAELRQRVQRLRNIVSQYETRY (SEQ ID NO:63) and LEIEAAFLEQENTALETEVAELEQEVQRLENIVSQYETRY (SEQ ID NO:64); EACGALEIRAAFLRRRNTALRTRVAELRQRVQRLRNIVSQYETRY (SEQ ID NO:65) and EACGALEIEAAFLEQENTALETEVAELEQEVQRLENIVSQYETRY (SEQ ID NO:66); EACGASTSVDELQAEVDQLEDENYALKTKVAQLRKKVEKL (SEQ ID NO:67) and EACGASTTVAQLEEKVKTLRAENYELKSEVQRLEEQVAQL (SEQ ID NO:68); AGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAH (SEQ ID NO:69) and AGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNY (SEQ ID NO:70);EACGAGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAH (SEQ ID NO:71) and EACGAGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNY (SEQ ID NO:72); GKIAALKQKIAALKYKNAALKKKIAALKQ (SEQ ID NO:73) and GEIAALEQEIAALEKENAALEWEIAALEQ (SEQ ID NO:74); and EACGAGKIAALKQKIAALKYKNAALKKKIAALKQ (SEQ ID NO:75) and EACGAGEIAALEQEIAALEKENAALEWEIAALEQ (SEQ ID NO:76). ;

[0163] In a preferred embodiment, the heterodimeric coiled-coil forming peptide is a sequence pair selected from SEQ ID NO: 45 and 46.

[0164] The homodimeric coiled-coil forming peptide can be, for example, GELEELLKHLKELLKGPRKGELEELLKHLKELLK (SEQ ID NO: 77).

[0165] The antibody hinge region is capable of forming disulfide bonds, thereby sterically interfering with the binding of the antibody to the antigen. The antibody hinge region can be a human antibody hinge region. The antibody hinge region can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. For example, the antibody hinge region can be derived from the hinge domain of human IgG1, IgG2, or IgG4, or a portion thereof. The amino acid sequence of the antibody hinge region can be selected from the group consisting of: EPKSCDKTHTCPPCP (SEQ ID NO: 78), EPKSCDKTHTCPPCPA (SEQ ID NO: 79), EPKSCDKTHTCPPCPAP (SEQ ID NO: 80), EPKSCDKTHTCPPCPAPE (SEQ ID NO: 81), EPKSCDKTHTCPPCPAPEL (SEQ ID NO: 82), EPKSCDKTHTCPPCPAPELL (SEQ ID NO: 83), EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 84), ERKCCVECPPCP (SEQ ID NO: 85), ERKCCVECPPCPA (SEQ ID NO: 86), ERKCCVECPPCPAP (SEQ ID NO: 87), ERKCCVECPPCPAPP (SEQ ID NO: 88), ERKCCVECPPCPAPPV (SEQ ID NO: 89), ERKCCVECPPCPAPPVA (SEQ ID NO: 90), ERKCCVECPPCPAPPVAG (SEQ ID NO: 91). In some embodiments, the antibody hinge region comprises the amino acid sequence of SEQ ID NO: 78.

[0166] Antibody fragments are able to target specific antigens, mask the antigen binding site within the antibody, and thereby sterically interfere with the binding of the antibody to the antigen. Examples of such antibody fragments include, for example, disulfide-stabilized variable fragments (dsFv) or intact antibodies.

[0167] The non-antibody protein fragment may be, for example, the LAP domain from TGF-β, which is capable of sterically interfering with the binding of the antibody to the antigen.

[0168] In some embodiments, the masking peptide inhibits the binding of the antibody to the antigen to which it is targeted by binding to the antibody's antigen binding site. Examples of such masking peptides include, but are not limited to, affinity polypeptides capable of binding to the antibody's antigen binding site and polypeptides comprising an antigenic epitope to which the antibody binds.

[0169] The affinity polypeptide capable of binding to the antigen binding site of the antibody may be a binding peptide from a bacterial peptide display library, which can specifically occupy the antigen binding site of the antibody and thus interfere with the binding of the antibody to the antigen.

[0170] A polypeptide comprising an antigenic epitope to which an antibody binds can mask the antigenic binding site of an antibody by binding to the antigenic binding site of the antibody, and may be, for example, an antigenic epitope, a polypeptide comprising an antigenic epitope, or a mutated antigen.

[0171] In a preferred embodiment, the masking peptide is selected from a coiled coil forming peptide and an antibody hinge region. In some embodiments, the VH1 and the VL1 are connected to a coiled coil forming peptide (e.g., a heterodimeric coiled coil forming peptide) at its N-terminus via a first linker, and the VH2 and the VL2 are connected to a coiled coil forming peptide (e.g., a heterodimeric coiled coil forming peptide) at its N-terminus via a second linker. In some embodiments, the VH1 and the VL1 are connected to an antibody hinge region via a first linker at its N-terminus, and the VH2 and the VL2 are connected to an antibody hinge region via a second linker at its N-terminus. In some embodiments, the VH1 and the VL1 are connected to a coiled coil forming peptide (e.g., a heterodimeric coiled coil forming peptide) at its N-terminus via a first linker, and the VH2 and the VL2 are connected to an antibody hinge region at its N-terminus via a second linker. In some embodiments, the VH1 and the VL1 are linked at their N-termini to an antibody hinge region via a first linker, and the VH2 and the VL2 are linked at their N-termini to a coiled-coil-forming peptide (e.g., a heterodimeric coiled-coil-forming peptide) via a second linker.

[0172] The antibodies disclosed herein can address LC / HC mispairing issues through CrossMAb technology. CrossMAb technology involves swapping a pair of HC and LC domains within the Fab region to resolve LC / HC mispairing, while retaining original antigen affinity. This is typically achieved in three ways: swapping VH and VL, swapping CH1 and CL, and swapping VH-CH1 and VL-CL.

[0173] In some embodiments, the VH and VL in the first Fab or the second Fab are replaced with each other. In some embodiments, the CH1 and CL in the first Fab or the second Fab are replaced with each other. In some embodiments, the VH and VL in the first Fab or the second Fab are replaced with each other and the CH1 and CL are replaced with each other.

[0174] In some embodiments, the antibody comprises:

[0175] A first heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a first linker, VH1, CH1, CH2, and CH3; and a first light chain comprising, from N-terminus to C-terminus, a masking peptide, a first linker, VL1, and CL; and a second heavy chain and a second light chain selected from:

[0176] (i) a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, CL, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, and CH1;

[0177] (ii) a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, CH1, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, and CL; or

[0178] (iii) a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, CL, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, and CH1.

[0179] In some embodiments, the antibody comprises: a first heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VH1, CL, CH2, and CH3; a first light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VL1, and CH1; a second heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, CH1, CH2, and CH3; and a second light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, and CL.

[0180] In some embodiments, the antibody comprises: a first heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VL1, CH1, CH2, and CH3; a first light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VH1, and CL; a second heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, CH1, CH2, and CH3; and a second light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, and CL.

[0181] In some embodiments, the antibody comprises: a first heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VL1, CL, CH2, and CH3; a first light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VH1, and CH1; a second heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, CH1, CH2, and CH3; and a second light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, and CL.

[0182] In some embodiments, the antibody comprises: a first heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VH1, CH1, CH2, and CH3; a first light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VL1, and CL; a second heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, CL, CH2, and CH3; and a second light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, and CH1.

[0183] In some embodiments, the antibody comprises: a first heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a first linker, VH1, CH1, CH2, and CH3; a first light chain comprising, from N-terminus to C-terminus, a masking peptide, a first linker, VL1, and CL; a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, CH1, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, and CL.

[0184] In some embodiments, the antibody comprises: a first heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VH1, CH1, CH2, and CH3; a first light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a first linker, VL, and CL; a second heavy chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, CL, CH2, and CH3; and a second light chain, which comprises, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, and CH1.

[0185] CH2 and CH3 constitute the Fc region of the antibody. The Fc region can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can contain one or more mutations or modifications. In one embodiment, the Fc region is of the IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0186] In some embodiments, the Fc region comprises modifications or mutations that can promote heterodimerization of the antibody heavy chains, such as a knob-to-hole mutation in CH3 of the Fc region.

[0187] A detailed description of the knob-hole concept can be found in, for example, U.S. Patent Nos. 5,731,168 and 7,186,076; and Ridgway et al., Protein Engineering, Design and Selection, 1996, 9(7):617–621, Atwell et al., J Mol Biol, 1997, 270(1):26-35; Merchant et al., Nat Biotechnol, 1998, 16:677-681; and Carter, J. Immunological Methods, 2001, 24(1-2):7-15. Briefly, a knob can be generated at the CH3 interface of a first IgG Fc chain by replacing a smaller one with a larger amino acid side chain (e.g., T366W); and a hole can be generated in juxtaposed positions at the CH3 interface of a second IgG Fc chain by replacing a larger one with a smaller amino acid side chain (e.g., Y407V).

[0188] The amino acid residues forming the knob are typically naturally occurring amino acid residues and are selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some preferred embodiments, the amino acid residues are tryptophan and tyrosine. In one embodiment, the original residues forming the knob have a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. Exemplary amino acid substitutions in the CH3 domain forming the knob include, but are not limited to, T366W, T366Y, or F405W substitutions.

[0189] The amino acid residues that form the hole are typically naturally occurring amino acid residues and are selected from alanine (A), serine (S), threonine (T), and valine (V). In some preferred embodiments, the original residues that form the hole have large side chain volumes, such as tyrosine, arginine, phenylalanine, or tryptophan. Exemplary amino acid substitutions in the CH3 domain that create the hole include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions.

[0190] In a preferred embodiment, the knob comprises a T366W substitution and the hole comprises T366S, L368A and Y407V substitutions.

[0191] In some embodiments, one or both of the CH3s contain at least one amino acid mutation capable of promoting heterodimerization of the first heavy chain and the second heavy chain; preferably, one of the CH3s contains T366S, L368A and Y407V mutations, and the other contains T366W mutation.

[0192] In some embodiments, the Fc region is defective in effector function. For example, the Fc region can be an IgG1 isotype, or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which has been mutated so that the ability to mediate effector functions such as ADCC is reduced or even eliminated. Such mutations have been described, for example, in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001). In some embodiments, the CH2 of the Fc region comprises at least one amino acid mutation that can reduce the effector function of the antibody, for example, selected from the following mutations: L234A, L235A, G237A, or any combination thereof.

[0193] In one embodiment, the Fc region comprises a mutation that removes an acceptor site for Asn-linked glycosylation or is otherwise manipulated to alter glycosylation properties. For example, in an IgG1 Fc region, an N297Q mutation can be used to remove an Asn-linked glycosylation site. Thus, in a specific embodiment, the Fc region comprises an IgG1 sequence having an N297Q mutation.

[0194] In a further embodiment, the Fc region is glycoengineered to reduce fucose and thereby enhance ADCC, for example by adding compounds to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, for example as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, one can use et al. (1999) Nature Biotech 17: 176 to optimize ADCC. In another embodiment, the Fc region is engineered to enhance complement activation, for example as described in Natsume et al. (2009) Cancer Sci. 100: 2411.

[0195] In some embodiments, CH1 and CL comprise amino acid mutations that promote dimerization of the heavy and light chains, such as mutations of positively charged amino acids (e.g., K and R) in one of CH1 and CL to negatively charged amino acids (e.g., E, Q, D, and N), and mutations of negatively charged amino acids in the other to positively charged amino acids. In a preferred embodiment, CH1 comprises K158E and K224E mutations, and CL comprises E143R and Q144K mutations (according to IMGT numbering).

[0196] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the first Fab and the second Fab bind to the same antigen. In some embodiments, the antigen is selected from tumor associated antigens (TAAs) and proinflammatory cytokines.

[0197] Many tumor-associated antigens (TAAs) associated with specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens that can potentially stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as antigens that are usually silent (i.e., not expressed) in normal cells, antigens that are only expressed at certain stages of differentiation, and antigens expressed over time, such as embryonic and fetal antigens. Other cancer cell antigens are encoded by mutant cell genes such as oncogenes (e.g., activated Ras oncogenes), suppressor genes (e.g., P53 mutants), and fusion proteins produced by internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes such as genes carried by RNA and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be prepared by those skilled in the art.

[0198] Examples of tumor-associated antigens include, but are not limited to, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, and combinations thereof.

[0199] Examples of proinflammatory cytokines include, but are not limited to, IL-1, IL-2, IL-6, IL-8, IL-12, TNF-α, IFN-γ, and macrophage migration inhibitory factor (MIF).

[0200] In some embodiments, the antigen is a TAA selected from the group consisting of CD20, EGFR, EpCAM, HER2, PSMA, gpA33, CD276, CEA, CD19, CD22, CD30, CD33, CD123, FLT3, and BCMA.

[0201] In some embodiments, the TAA is CD20, and wherein VH1 and VH2 comprise HCDRs 1-3 having the following amino acid sequences, respectively: GYTFTSYN (SEQ ID NO: 17), IYPGNGDT (SEQ ID NO: 18), and AR, and VL1 and VL2 comprise LCDRs 1-3 having the following amino acid sequences, respectively: SSVSY (SEQ ID NO: 20), ATS, and QQWTSNP (SEQ ID NO: 21).

[0202] In some embodiments, VH1 and VH2 comprise the amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19, and the VL1 and VL2 comprise the amino acid sequence as set forth in SEQ ID NO: 22, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22.

[0203] In some embodiments, VH1 and VH2 comprise functional variants of the amino acid sequence as shown in SEQ ID NO: 19, which are formed by inserting, deleting and / or substituting one or more amino acids therein, and the precursor is that the functional variant retains the ability to bind to the antigen. In some embodiments, VL1 and VL2 comprise functional variants of the amino acid sequence as shown in SEQ ID NO: 22, which are formed by inserting, deleting and / or substituting one or more amino acids therein, and the precursor is that the functional variant retains the ability to bind to the antigen.

[0204] In some embodiments, the number of inserted, deleted and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, and more preferably 15% to 20%. For example, the number of inserted, deleted and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0205] In some embodiments, insertions, deletions and / or substitutions may be made in the framework (FR) regions, eg, in FR1, FR2, FR3 and / or FR4.

[0206] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is substituted with another amino acid residue in the same group. (a) Small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) Polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) Polar, positively charged residues: His, Arg and Lys; (d) Large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; and (e) Aromatic residues: Phe, Tyr and Trp.

[0207] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0208] In a preferred embodiment, VH1 and VH2 comprise the amino acid sequence shown in SEQ ID NO: 19 and VL1 and VL2 comprise the amino acid sequence shown in SEQ ID NO: 22.

[0209] In some embodiments, the antibody further comprises a signal peptide sequence at the N-terminus of each chain, such as a signal peptide sequence derived from albumin or immunoglobulin, preferably MHSSALLCCLVLLTGVRA (SEQ ID NO: 100).

[0210] In some embodiments, the antibody comprises: a first heavy chain comprising amino acids as set forth in SEQ ID NO:5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5; a second heavy chain comprising amino acids as set forth in SEQ ID NO:6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:6; a first light chain comprising amino acids as set forth in SEQ ID NO:7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:7; and a second light chain comprising amino acids as set forth in SEQ ID NO:8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:8 (Molecule 2).

[0211] In some embodiments, the antibody comprises: a first heavy chain comprising amino acids as set forth in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:9; a second heavy chain comprising amino acids as set forth in SEQ ID NO:10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:10; a first light chain comprising amino acids as set forth in SEQ ID NO:11, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:11; and a second light chain comprising amino acids as set forth in SEQ ID NO:12, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:12. NO:12 has an amino acid sequence (molecule 3) with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0212] In some embodiments, the antibody comprises: a first heavy chain comprising amino acids as set forth in SEQ ID NO: 13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13; a second heavy chain comprising amino acids as set forth in SEQ ID NO: 14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14; a first light chain comprising amino acids as set forth in SEQ ID NO: 15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15; and a second light chain comprising amino acids as set forth in SEQ ID NO: 16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16. NO:16 has an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity (molecule 4).

[0213] In some embodiments, the first heavy chain comprises a functional variant of the amino acid sequence as shown in any one of SEQ ID NOs: 5, 9, and 13, which is formed by insertion, deletion, and / or substitution of one or more amino acids, provided that the functional variant retains the ability to bind to the antigen. In some embodiments, the second heavy chain comprises a functional variant of the amino acid sequence as shown in any one of SEQ ID NOs: 6, 10, and 14, which is formed by insertion, deletion, and / or substitution of one or more amino acids, provided that the functional variant retains the ability to bind to the antigen. In some embodiments, the first light chain comprises a functional variant of the amino acid sequence as shown in any one of SEQ ID NOs: 7, 11, and 15, which is formed by insertion, deletion, and / or substitution of one or more amino acids, provided that the functional variant retains the ability to bind to the antigen. In some embodiments, the second light chain comprises a functional variant of the amino acid sequence as shown in any one of SEQ ID NOs: 8, 12, and 16, which is formed by insertion, deletion, and / or substitution of one or more amino acids, provided that the functional variant retains the ability to bind to the antigen.

[0214] In some embodiments, the number of inserted, deleted and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, and more preferably 15% to 20%. For example, the number of inserted, deleted and / or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0215] In some embodiments, insertions, deletions and / or substitutions may be made in the framework (FR) regions, e.g., FR1, FR2, FR3 and / or FR4; and / or constant regions, e.g., CL, CH1, CH2 and / or CH3.

[0216] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0217] The Double-Lock antibodies disclosed herein are also applicable to bispecific antibodies. Bispecific antibodies can simultaneously recognize two distinct epitopes on the same or different antigens, overcoming the shortcomings of traditional monoclonal antibodies and improving their efficacy. They can be applied in a variety of therapeutic areas, such as cancer, chronic inflammatory diseases, autoimmune diseases, and infections.

[0218] The main technical difficulty in producing bispecific antibodies is obtaining correctly paired bispecific antibodies. Currently, a variety of platform technologies have been developed to address the mispairing problem of bispecific antibodies, such as Triomab quadroma, KiH (Knob-into-hole), CrossMab, ART-lg, BiMab, and DuoBody.

[0219] Triomab quadroma technology is a bispecific antibody obtained by somatic cell hybridization of CD3-specific rat IgG2b antibody and tumor-targeting mouse IgG2a antibody. It was jointly developed by Fresenius and Trion Pharma and solves the HC / HC and LC / HC mismatch problems.

[0220] KiH technology, which engineers HC to create heterodimers, represents a breakthrough in resolving the HC / HC problem. KiH technology introduces mutations in the CH3 region of one heavy chain, creating a protruding "knob"-like structure, while mutations in the CH3 region of the other heavy chain create a recessed "hole" structure. This knob-and-hole design facilitates the proper assembly of the two heterologous antibody heavy chains.

[0221] CrossMAb technology, developed by Roche, involves swapping the HC and LC domains within the Fab region to resolve LC / HC mispairing. This swapping technique preserves the original antigen affinity. This is typically achieved in three ways: swapping VHb and VLb, swapping CH1b and CLb, and swapping VHb-CH1b and VLb-CLb.

[0222] Both ART-lg and BiMab technologies use electrostatic steering mutations to solve the HC / HC mismatch problem, that is, introducing negatively charged and positively charged amino acid residues into the CH3 domains of two different heavy chains, respectively, to increase the formation of heterodimers through electrostatic attraction and reduce the formation of homodimers through electrostatic repulsion.

[0223] Developed by Genmab, the DuoBody platform technology forms half-antibodies or bispecific antibodies based on the naturally occurring Fab arm exchange mechanism of IgG4 antibodies in the human body. The technology involves first expressing two IgG1 antibodies separately, introducing mutations into the CH3 regions of each antibody to address HC / HC mispairing. The two target antibodies are then mixed, reacted with a reducing agent (such as GSH) to form half-antibodies, and then reassembled into an IgG1 bispecific antibody.

[0224] For the bispecific antibodies of the present invention, the first Fab and the second Fab bind to different antigens, and the first Fab and the second Fab have high binding affinity to ensure monovalent binding activity. In some embodiments, the first Fab and the second Fab have a KD value of at least 10 -8 In some embodiments, the first Fab and the second Fab have a binding affinity of at least 10 M as measured by a KD value. -9 In some embodiments, the first Fab and the second Fab have a binding affinity of at least 10 M as measured by a KD value. -10 In some embodiments, the first Fab and the second Fab have a binding affinity of at least 10 M as measured by a KD value. -11 In some embodiments, the first Fab and the second Fab have a binding affinity of at least 10 M as measured by a KD value. -12 The binding affinity of M.

[0225] In some embodiments of the bispecific antibody, one of the antigens is selected from the group consisting of a TAA and a proinflammatory cytokine, and the other is selected from the group consisting of a TAA, a proinflammatory cytokine, and an immune cell antigen.

[0226] Examples of TAA and proinflammatory cytokines are as described above. Immune cell antigens can be, for example, T cell antigens. T cell antigens can be selected from T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) and NKG2D.

[0227] In another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody disclosed herein.

[0228] In yet another aspect, the present invention provides a vector comprising a nucleic acid disclosed herein.

[0229] Any vector may be suitable for use in the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pBY, pGAR, pBABE-puro, pBABE-neo largeT cDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0230] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include vectors designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, vectors can be selected from pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors that can be used in the context of the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0231] Recombinant expression vectors can be prepared using standard recombinant DNA techniques. Circular or linear expression vector constructs can be prepared to contain replication systems functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, CO1E1, 2μ plasmids, lambda, SV40, bovine papilloma virus, etc.

[0232] In yet another aspect, the invention provides a host cell comprising a nucleic acid or a vector disclosed herein.

[0233] Any cell can be used as the host cell of nucleic acid or vector of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include but are not limited to true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae (Enterobactehaceae), such as Escherichia (Escherichia), such as Escherichia coli (E.coli); Enterobacter (Enterobacter); Erwinia (Erwinia); Klebsiella (Klebsiella); Proteus (Proteus); Salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); Serratia (Serratia), such as Serratia marcescens (Serratia marcescens). marcescans and Shigella; Bacilli, such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0234] In yet another aspect, the present invention provides a pharmaceutical composition comprising (i) an antibody disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0235] In some embodiments, carriers or excipients used with the compositions disclosed herein include, but are not limited to, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and the surfactant polyoxyethylene-sorbitan monooleate.

[0236] In some embodiments of the pharmaceutical composition disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent.

[0237] In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor.In some embodiments, the second therapeutic agent is a chemotherapeutic agent.Chemotherapeutic agents can include, for example, cytotoxic agents, antimetabolites (such as folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (such as camptothecin derivatives, anthraquinones, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.), antimicrotubule agents (such as taxanes, vinca alkaloids), protein synthesis inhibitors (such as cephalotaxine, camptothecin derivatives, quinoline alkaloids), alkylating agents (such as alkyl sulfonates, aziridine rings, nitrogen mustards, nitrosoureas, platinum derivatives, triazene, etc.), alkaloids, terpenoids and kinase inhibitors.

[0238] In yet another aspect, the present invention provides a conjugate comprising an antibody disclosed herein and a chemical moiety conjugated thereto.

[0239] In some embodiments of the presently disclosed conjugates, the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

[0240] In some embodiments, therapeutic agents include but are not limited to immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins. In some embodiments, therapeutic agents can be, for example, maytansine, geldanamycin, tubulin inhibitors such as tubulin binders (e.g., auristatins), or minor groove binders such as calicheamicin.

[0241] Other suitable therapeutic agents include, for example, small molecule cytotoxic agents, i.e., compounds with a molecular weight of less than 700 Daltons that have the ability to kill mammalian cells. These compounds may also contain toxic metals that can have cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decompose or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, razithromycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, metformin, auristatin E, vincristine, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes, and RNA enzymes; radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225, and astatine-213; chelating agents can be used to facilitate the binding of these radionuclides to molecules or polymers thereof.

[0242] In some embodiments, the detectable moiety can be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

[0243] In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule can be a cytokine such as IL-2 and IFN-γ, a chemokine such as IL-8, platelet factor 4, melanoma growth stimulating protein, a complement activator; a viral / bacterial protein domain, or a viral / bacterial peptide.

[0244] In yet another aspect, the present invention provides a method of treating a disease in a subject, comprising administering to the subject an effective amount of an antibody disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein.

[0245] In some embodiments of the methods disclosed herein, the disease is cancer or an inflammatory disease.

[0246] The cancer can be any cancer, such as a hematologic cancer, a central and peripheral nervous system cancer, a lymphoid cancer, a myeloid cancer, a mesenchymal-derived cancer, a solid tumor, and the like.

[0247] In some embodiments, the TAA is CD20 and the cancer is a CD20-positive cancer, such as a lymphoma.

[0248] In some embodiments, the cancer is cervical cancer, lung cancer, liver cancer, breast cancer, and colon cancer.

[0249] In some embodiments, the inflammatory disease is selected from rheumatoid arthritis, drug-induced hepatitis, liver fibrosis, chronic obstructive pulmonary disease, asthma, and atopic dermatitis.

[0250] In some embodiments, the dosage administered to a subject may vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine the effective amount to administer to a human or other subject to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as the activity of the antibody and the route of administration.

[0251] The dosage of the antibodies, compositions or conjugates described herein can be administered to a mammal at once or in a series of sub-doses over an appropriate time period, for example, daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annually or annually as needed. A dosage unit comprising an effective amount of the antibody, composition or conjugate can be administered in a single daily dose, or the total daily dose can be administered in two, three, four or more divided doses administered daily as needed.

[0252] Suitable modes of administration can be selected by a physician. The route of administration can be parenteral administration, for example, by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition or conjugate is selected for parenteral delivery, for inhalation or for delivery through the digestive tract, for example, orally. The dosage and method of administration can vary according to the weight, age, condition, etc. of the subject and can be appropriately selected.

[0253] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.In certain embodiments, the antibodies, compositions, or conjugates disclosed herein are administered prior to, substantially simultaneously with, or after administration of the second therapeutic agent.

[0254] In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0255] In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor.In some embodiments, the second therapeutic agent is a chemotherapeutic agent.Chemotherapeutic agents can include, for example, cytotoxic agents, antimetabolites (such as folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (such as camptothecin derivatives, anthraquinones, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.), antimicrotubule agents (such as taxanes, vinca alkaloids), protein synthesis inhibitors (such as cephalotaxine, camptothecin derivatives, quinoline alkaloids), alkylating agents (such as alkyl sulfonates, aziridine rings, nitrogen mustards, nitrosoureas, platinum derivatives, triazene, etc.), alkaloids, terpenoids and kinase inhibitors.

[0256] In another aspect, the invention provides a pharmaceutical package or kit comprising one or more containers containing one or more components of the pharmaceutical compositions described herein, e.g., antibodies disclosed herein. Optionally, associated with such containers may be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.

[0257] In a specific embodiment, the kit comprises a first container containing an antibody disclosed herein. In a specific embodiment, the kit comprises a first container that is a vial containing the antibody as a lyophilized sterile powder under vacuum, and the kit further comprises a second container containing a pharmaceutically acceptable fluid.

[0258] In certain embodiments, provided herein are injection devices comprising an antibody. In certain embodiments, the injection device comprises an antibody in a sterile solution. In certain embodiments, the injection device is a syringe.

[0259] In another aspect, the present invention provides a use of an antibody disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein in the preparation of a medicament for treating a disease in a subject. In some embodiments, an antibody disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein is combined with a second therapeutic agent.

[0260] In another aspect, the present invention provides an antibody disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein for use in treating a disease in a subject.

[0261] In some embodiments of the uses disclosed herein, the disease is cancer or an inflammatory disease.

[0262] The cancer can be any cancer, such as hematologic cancer, central and peripheral nervous system cancer, lymphoid cancer, myeloid cancer, mesenchymal-derived cancer, solid tumors, etc. In some embodiments, the cancer is selected from cervical cancer, lung cancer, liver cancer, breast cancer, and colon cancer.

[0263] In some embodiments, the inflammatory disease is selected from rheumatoid arthritis, drug-induced hepatitis, liver fibrosis, chronic obstructive pulmonary disease, asthma, and atopic dermatitis.

[0264] In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug. In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor.

[0265] Example

[0266] The following examples are provided for the purpose of illustrating various embodiments of the present invention, but are not intended to limit the present invention in any way. The present examples and the methods described herein currently represent preferred embodiments and are exemplary and are not intended to limit the scope of the present invention. Those skilled in the art will appreciate variations and other uses that are encompassed within the spirit of the present invention as defined by the scope of the claims.

[0267] Example 1: Antibody molecule design

[0268] Three protease-cleavable double-locked antibody molecules were constructed, designated molecules 2, 3, and 4. These molecules are cleaved by MMP2, MMP9, and uPA to become molecule 1. Schematic diagrams of the structures of molecules 1-4 are shown in Figures 1-4. The sequence information of molecules 1-4 is shown below.

[0269] Molecule 1

[0270] Heavy chain H1 sequence: SEQ ID NO: 1, structure CD20 VH-CH1-Fc (human IgG1 hole mutation)

[0271] Heavy chain H2 sequence: SEQ ID NO: 2, its structure is CD20 VH-CL (human κ chain)-Fc (human IgG1 knob mutation)

[0272] Light chain L1 sequence: SEQ ID NO: 3, its structure is CD20 VL-CL (human κ chain)

[0273] Light chain L2 sequence: SEQ ID NO: 4, its structure is CD20 VL-CH1 (human IgG1)

[0274] Molecule 2

[0275] Heavy chain H1 sequence: SEQ ID NO: 5, its structure is masked peptide-protease cleavable linker-CD20 VH-CH1-Fc (human IgG1 hole mutation)

[0276] Heavy chain H2 sequence: SEQ ID NO: 6, its structure is masked peptide-protease cleavable linker-CD20 VH-CL (human kappa chain)-Fc (human IgG1 knob mutation)

[0277] Light chain L1 sequence: SEQ ID NO: 7, its structure is masked peptide-protease cleavable linker-CD20 VL-CL (human κ chain)

[0278] Light chain L2 sequence: SEQ ID NO: 8, its structure is masked peptide-protease cleavable linker-CD20 VL-CH1 (human IgG1)

[0279] Molecule 3

[0280] Heavy chain H1 sequence: SEQ ID NO: 09, its structure is masked peptide-protease cleavable linker-CD20 VH-CH1-Fc (human IgG1 hole mutation)

[0281] Heavy chain H2 sequence: SEQ ID NO: 10, its structure is masked peptide-protease cleavable linker-CD20 VH-CL (human kappa chain)-Fc (human IgG1 knob mutation)

[0282] Light chain L1 sequence: SEQ ID NO: 11, its structure is masked peptide-protease cleavable linker-CD20 VL-CL (human κ chain)

[0283] Light chain L2 sequence: SEQ ID NO: 12, its structure is masked peptide-protease cleavable linker-CD20 VL-CH1 (human IgG1)

[0284] Molecule 4

[0285] Heavy chain H1 sequence: SEQ ID NO: 13, its structure is masked peptide-protease cleavable linker-CD20 VH-CH1-Fc (human IgG1 hole mutation)

[0286] Heavy chain H2 sequence: SEQ ID NO: 14, its structure is masked peptide-protease cleavable linker-CD20 VH-CL (human kappa chain)-Fc (human IgG1 knob mutation)

[0287] Light chain L1 sequence: SEQ ID NO: 15, its structure is masked peptide-protease cleavable linker-CD20 VL-CL (human κ chain)

[0288] Light chain L2 sequence: SEQ ID NO: 16, its structure is masked peptide-protease cleavable linker-CD20 VL-CH1 (human IgG1)

[0289] In the above molecules 1-4, the masking peptide sequence is shown in italics, the protease cleavage site is underlined, and the antibody variable regions VH and VL are shown in bold. The amino acid sequences of VH and VL, which bind to CD20, and the CDR sequences therein (according to the IMGT numbering system) are shown below.

[0290] VH:

[0291] HCDR1:

[0292] HCDR2:

[0293] HCDR3:

[0294] VL:

[0295] LCDR1:

[0296] LCDR2:

[0297] LCDR3:

[0298] To increase the secretion of the antibody molecule, the signal peptide sequence MHSSALLCCLVLLTGVRA (SEQ ID NO: 100) was added to the N-terminus of each chain of molecules 1-4.

[0299] Example 2: Expression, purification and characterization of antibody molecules

[0300] Antibody molecules 1-4 were prepared and characterized as follows.

[0301] 1. Experimental Methods

[0302] Expression system: ExpiCHO TM Expression System (Thermo, A29133).

[0303] Expression cell line: ExpiCHO-S cells.

[0304] Expression conditions: 125 mL shake flask, 120 rpm, 37°C, 8% carbon dioxide.

[0305] Purification method: Protein A gel-bound antibody Fc (Biyuntian, P2015-50ML).

[0306] 2. Expression steps:

[0307] 2.1. Cell preparation:

[0308] Following the ExpiCHO kit protocol, passage and expand ExpiCHO-S cells until the cell density reaches approximately 4-6 x 10 6 cells / mL and dilute the cells in culture medium to a density of approximately 3–4 x 10 6 The cells were cultured in an incubator overnight.

[0309] 2.2. Antibody plasmid transfection:

[0310] The cell density should reach approximately 7-10 x 10 6 cells / mL, the viability reached more than 95%; the cell density was diluted with culture medium to about 6 x 10 6 cells / mL, and an expression volume of 25 mL per molecule. Plasmid dosages were as follows: 13.3 μg of molecule 1-4 light chain plasmid and 6.67 μg of molecule 1-4 heavy chain plasmid. TM Dilute the plasmid DNA to 1 mL with cold culture medium and take OptiPRO TM 920 μl of culture medium with ExpiFectamine TM Mix 80μl of CHO reagent; dilute ExpiFectamine TM Add CHO reagent to the diluted DNA and let it stand at room temperature for 1-5 minutes. Slowly transfer the mixed solution to a shake flask and culture it in an incubator at 37°C, 8% carbon dioxide, and 120 rpm.

[0311] 2.3. Adding feed

[0312] 18-22 hours after transfection, add 150 μl ExpiFectamine CHO Enhancer and 4 ml ExpiCHO to each flask. TM Feed.

[0313] 2.4. Collecting the supernatant

[0314] The supernatant was collected 6-8 days after transfection, centrifuged at 4000 g for 30 min, and the supernatant was collected and stored at 4°C.

[0315] 3. Protein Purification

[0316] 3.1. Take 2 ml of Protein A gel into a 50 ml centrifuge tube, wash twice with 10 ml of PBS, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0317] 3.2. Add cell supernatant to Protein A gel and incubate on a rotary mixer at 50 rpm for 3 h at room temperature.

[0318] 3.3. Wash the Protein A gel five times with 10 ml of PBS and centrifuge at 2000 rpm for 5 minutes.

[0319] 3.4. Elute the Protein A gel three times with 2 ml of sodium acetate eluent, and add TRIS-HCl, pH 9.0 neutralizer until the pH of the eluent is between 7.0 and 8.0.

[0320] 3.5. Place the antibody solution at 4°C and prepare for analysis.

[0321] 4. SDS-PAGE and SEC-HPLC Characterization

[0322] The purified molecules 1 to 4 were subjected to SDS-PAGE, and the results are shown in Figures 5, 7, 9, and 11, respectively.

[0323] The purified molecules 1 to 4 were subjected to SEC-HPLC, and the results are shown in Figures 6, 8, 10, 12 and Tables 1-4 below, respectively.

[0324] Table 1. SEC-HPLC parameters of molecule 1

[0325] Detector A channel 1 214 nm

[0326] Detector A channel 2 280nm

[0327] Table 2. SEC-HPLC profile parameters of molecule 2

[0328] Detector A channel 1 214 nm

[0329] Detector A channel 2 280nm

[0330] Table 3. SEC-HPLC profile parameters of molecule 3

[0331] Detector A channel 1 214 nm

[0332] Detector A channel 2 280nm

[0333] Table 4. SEC-HPLC profile parameters of molecule 4

[0334] Detector A channel 1 214 nm

[0335] Detector A channel 2 280nm

[0336] Example 3: Enzyme Activation and Enzyme Cleavage of Masked Peptide Antibodies

[0337] 1. Enzymes used

[0338] Urokinase (abbreviation: uPA, R&D, catalog number: 1310-SE), recombinant human metalloproteinase 2 (abbreviation: MMP-2, R&D, catalog number: 902-MP), recombinant human metalloproteinase 9 (abbreviation: MMP-9, R&D, catalog number: 911-MP).

[0339] 2. Reagents

[0340] Enzyme activation reagent: p-Aminophenylmercuric acetate AMPA (Sigma, product number: A-9563).

[0341] Metalloproteinase activation buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij35, pH = 7.5.

[0342] 3. Protease Activation Step

[0343] 3.1. Activation of MMP-9 enzyme: According to the manufacturer's instructions, the enzyme was mixed with AMPA and metalloproteinase buffer as shown in the table below, and incubated in a 37°C water bath for 24 hours. After activation, the concentration of MMP-9 enzyme was 0.66 μM.

[0344] Table 5. Preparation of mixed solution for activating MMP-9 enzyme

[0345] 3.2. Activation of MMP-2 enzyme: According to the manufacturer's instructions, the enzyme was mixed with AMPA and metalloproteinase buffer as shown in the table below, and incubated in a 37°C water bath for 1 hour. After activation, the concentration of MMP-2 enzyme was 0.71 uM.

[0346] Table 6. Formulation of MMP-2 enzyme

[0347] 3.3. uPA enzyme does not need to be activated, just dilute it: the initial stock solution concentration is 5.23 μM, take 3.8 μl uPA enzyme supplemented with metalloproteinase buffer and dilute it to 20 μl, which is 1 μM.

[0348] 3.4. Enzymatic cleavage reaction conditions of the three activating enzymes with antibody molecules 2, 3, and 4

[0349] In a 37°C water bath, uPA enzyme was first added and digested for 2 hours. MMP-2 enzyme was then added and digested for 1 hour. Finally, MMP-9 enzyme was added and digested for 18 hours. The molar ratio of antibody to each enzyme was 10:1. The concentration of molecules 2, 3, and 4 after triple digestion was 1.28 μM.

[0350] Table 7. Enzyme digestion conditions for molecules 2, 3, and 4

[0351] Example 4: Cell Binding Activity of Antibody Molecules Before and After Enzymatic Digestion

[0352] Using molecule 1 as a positive control, the binding activity of molecules 2-4 before and after enzyme digestion to CD20-positive lymphoma Raji cells was compared.

[0353] 1. Antibody Dilution

[0354] The antibody was diluted 3-fold using 1640 medium, with the highest concentration being 100 nM. Eight gradients were set for 3-fold dilution.

[0355] 2. FACS Binding Experiment

[0356] Take 2×10 7 After centrifugation, Raji cells were resuspended in 2 mL FACS buffer and Fc receptors on Raji cells were blocked with 50 μl Human TruStain FcX (Biolegend, Cat. No. 422302) for 10 min. FACS buffer was added to the Raji cell suspension to make 5 ml, and 50 μl of Raji cell suspension was added to each well of a 96-well plate, i.e., 2 × 10 cells per well. 5 For each well, 100 μl of diluted antibody was added and incubated at 4°C for 30 min. The cells were then washed three times with FACS buffer, followed by the addition of 2 μl of goat anti-human IgG Fc secondary antibody, PE (Thermo Fisher, Cat. No. 12-4998-82), and incubation at 4°C for 30 min. After washing the cells three times with FACS buffer, the cells were resuspended in 150 μl of FACS buffer per well and loaded for analysis. Data were then processed and the median values ​​of the PE fluorescence channel were analyzed. The median values ​​were imported into GraphPad software, and the results are shown in Figures 13-15 and Tables 8-10 below.

[0357] Table 8. FACS binding assay results for molecule 2

[0358] Table 9. FACS binding assay results for molecule 3

[0359] Table 10. FACS binding assay results for molecule 4

[0360] The results of Figure 13 and Table 8 show that after molecule 2 is digested, the EC50 and Top value (the upper platform value of the FACS MFI value curve fitting) are close to those of molecule 1, indicating that the triple enzyme digestion system completely restores the binding activity of molecule 2 to Raji cells. The results of Figure 14 and Table 9 show that after molecule 3 is digested, the EC50 is approximately 1.5 times that of molecule 1, and the Top value is close to that of molecule 1, indicating that the triple enzyme digestion system completely restores the binding activity of molecule 3 to Raji cells. The results of Figure 15 and Table 10 show that after molecule 4 is digested, the EC50 is approximately 2.3 times that of molecule 1, indicating that the triple enzyme digestion system restores the binding activity of molecule 4 to Raji cells.

[0361] Example 5: Comparison of ADCC activity of molecule 4 before and after enzyme cleavage on Raji cells using EuTDA method

[0362] Recovery 3×10 7 PBMC (Shanghai Aoneng Biotechnology Co., Ltd.) was resuspended in 10 ml of complete medium (1640 + 10% FBS) and incubated overnight. Raji cells were washed 6 times with PBS containing 20 mM HEPES and 2 mM probenecid, and the Raji cell pellet was centrifuged at 1200 rpm for 5 min. Raji cells were resuspended in 1640 medium containing 2 mM probenecid and 10% FBS, and the cell density was adjusted to 1 × 10 5 cells / ml; resuspend PBMC in 1640 medium containing 2 mM probenecid and 10% FBS to adjust the density to 2.5 × 10 6 Cells / ml; dilute the antibody to a maximum concentration of 20 nM using 1640 medium, and perform a 3-fold serial dilution, for a total of seven steps. Lysis, blank, and experimental groups were set up and added to 96-well plates. The lysis group consisted of 100 μl target cells, 10 μl lysis buffer, and 90 μl complete medium. The blank group consisted of 100 μl target cells, 80 μl PBMC, and 20 μl complete medium. The experimental group consisted of 100 μl target cells, 80 μl PBMC, and 20 μl antibody.

[0363] The plate was incubated in a 37°C, 5% CO2 incubator for 4 hours. Cells were pelleted by centrifugation at 1800 rpm for 10 minutes. 20 μl of supernatant was added to 200 μl of Eu solution. Microplate reader was used for detection with an excitation wavelength of 340 nm, an emission wavelength of 615 nm, a delay of 400 μs, and a window of 400 μs. Cytotoxicity was calculated as follows: % cytotoxicity = (antibody-treated group - no antibody group) / (lysis group - no antibody group) × 100. The results are shown in Figure 16 and Table 11 below.

[0364] Table 11. Cytotoxicity test results

[0365] The results in Figure 16 and Table 11 show that after triple enzyme digestion, the ADCC activity of molecule 4 was 50 It is 2.3 times that of molecule 1, which is consistent with the binding activity of molecule 4 to Raji cells after triple enzyme digestion (the multiple is also 2.3 times), indicating that the in vitro activity of the antibody is basically restored after the antibody masking peptide is digested.

[0366] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments described herein may be employed. The following claims are intended to define the scope of the invention and encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. A protease-cleavable antibody comprising: a first Fab comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and a second Fab comprising a second heavy chain variable region (VH2) and a second light chain variable region (VL2); wherein said VH1 and / or said VL1 are linked to a masking peptide via a first linker comprising a first protease cleavage site; wherein said VH2 and / or said VL2 are linked to the masking peptide via a second linker comprising a second protease cleavage site; The first protease cleavage site is specifically cleaved by a first protease, the second protease cleavage site is specifically cleaved by a second protease, and the first protease and the second protease are different. 2 . The antibody according to claim 1 , wherein the VH1 and / or the VL1 are linked at their N-termini to the masking peptide via the first linker, and the VH2 and / or the VL2 are linked at their N-termini to the masking peptide via the second linker. 3 . The antibody according to claim 2 , wherein the VH1 and the VL1 are linked to the masking peptide at their N-termini via the first linker, and the VH2 and the VL2 are linked to the masking peptide at their N-termini via the second linker.

4. The antibody according to any one of claims 1-3, wherein the first protease and the second protease are each independently a protease expressed or overexpressed in a tumor microenvironment or an inflammatory environment.

5. The antibody of any one of claims 1-4, wherein the first protease and the second protease are each independently selected from the group consisting of serine proteases, metalloproteases, thiol proteases, and carboxyl proteases.

6. The antibody of any one of claims 1-5, wherein the first protease and the second protease are each independently selected from uPA, MMP, TEV protease, plasmin, thrombin, FXa, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM, ADAMTS, caspases, enterokinase, and HRV 3C protease.

7. The antibody according to any one of claims 1 to 5, wherein one of the first protease and the second protease is a serine protease and the other is a metalloprotease.

8. The antibody according to claim 7, wherein one of the first protease and the second protease is uPA and the other is a MMP (eg, selected from MMP2 and MMP9).

9. The antibody according to any one of claims 1 to 8, wherein the first protease cleavage site and the second protease cleavage site are each independently selected from SEQ ID NOs: 23 to 32, preferably selected from SEQ ID NO: 23 and SEQ ID NO:

24.

10. The antibody of any one of claims 1-9, wherein the first linker and the second linker each independently comprise an amino acid sequence selected from SEQ ID NOs: 23-42.

11. The antibody according to any one of claims 1 to 10, wherein the masking peptide is capable of inhibiting the binding of the antibody to the antigen to which it is targeted and has a length of 5 to 60 amino acids.

12. The antibody according to any one of claims 1 to 11, wherein the masking peptide inhibits the binding of the antibody to the antigen to which it is targeted by steric hindrance and / or binding to the antigen binding site of the antibody.

13. The antibody according to any one of claims 1 to 12, wherein the masking peptide is selected from the group consisting of a coiled-coil forming peptide, an antibody hinge region, an antibody fragment (e.g., dsFv), a non-antibody protein fragment (e.g., the LAP domain from TGF-β), an affinity polypeptide capable of binding to the antigen binding site of the antibody, and a polypeptide comprising an antigenic epitope to which the antibody binds, preferably selected from the group consisting of a coiled-coil forming peptide and an antibody hinge region.

14. The antibody of claim 13, wherein the coiled-coil-forming peptide is a heterodimeric coiled-coil-forming peptide (e.g., a sequence pair selected from the group consisting of SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54) or a homodimeric coiled-coil-forming peptide (e.g., SEQ ID NO: 77).

15. The antibody according to claim 13, wherein the antibody hinge region is a human antibody hinge region, eg, comprising the amino acid sequence of SEQ ID NO:

78.

16. The antibody according to any one of claims 1 to 15, wherein: The VH and VL in the first Fab or the second Fab are replaced with each other, CH1 and CL in the first Fab or the second Fab are replaced with each other, or In the first Fab or the second Fab, VH and VL are replaced with each other, and CH1 and CL are replaced with each other.

17. The antibody of claim 16, wherein the antibody comprises: A first heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a first linker, VH1, CH1, CH2, and CH3; and a first light chain comprising, from N-terminus to C-terminus, a masking peptide, a first linker, VL1, and CL; and a second heavy chain and a second light chain selected from: (i) a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, CL, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, and CH1; (ii) a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, CH1, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, and CL; or (iii) a second heavy chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VL2, CL, CH2, and CH3; and a second light chain comprising, from N-terminus to C-terminus, a masking peptide, a second linker, VH2, and CH1.

18. The antibody of claim 17, wherein one or both of the CH3s comprise at least one amino acid mutation capable of promoting heterodimerization of the first heavy chain and the second heavy chain; Preferably, one of the CH3 comprises T366S, L368A and Y407V mutations, and the other comprises T366W mutation.

19. The antibody of any one of claims 1-18, wherein the first Fab and the second Fab bind to the same antigen.

20. The antibody of claim 19, wherein the antigen is selected from the group consisting of a tumor-associated antigen (TAA) and a pro-inflammatory cytokine.

21. The antibody of any one of claims 1-18, wherein the first Fab and the second Fab bind to different antigens, and the first Fab and the second Fab have KD values ​​that are at least 10 -8 The binding affinity of M.

22. The antibody of claim 21, wherein one of the antigens is selected from the group consisting of TAAs and proinflammatory cytokines, and the other is selected from the group consisting of TAAs, proinflammatory cytokines, and immune cell antigens (eg, T cell antigens).

23. The antibody of claim 20, wherein the TAA is CD20, and wherein The VH1 and VH2 comprise HCDRs 1-3 having the following amino acid sequences, respectively: GYTFTSYN (SEQ ID NO: 17), IYPGNGDT (SEQ ID NO: 18), and AR, and the VL1 and VL2 comprise LCDRs 1-3 having the following amino acid sequences, respectively: SSVSY (SEQ ID NO: 20), ATS, and QQWTSNP (SEQ ID NO: 21).

24. The antibody of claim 23, wherein the VH1 and VH2 comprise the amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and the VL1 and VL2 comprise the amino acid sequence as set forth in SEQ ID NO: 22, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

25. The antibody of claim 1, wherein the antibody comprises: (i) a first heavy chain comprising amino acids as set forth in SEQ ID NO:5, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:5; a second heavy chain comprising amino acids as set forth in SEQ ID NO:6, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:6; a first light chain comprising amino acids as set forth in SEQ ID NO:7, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:7; and a second light chain comprising amino acids as set forth in SEQ ID NO:8, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:8; or (ii) a first heavy chain comprising amino acids as set forth in SEQ ID NO:9, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:9; a second heavy chain comprising amino acids as set forth in SEQ ID NO:10, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:10; a first light chain comprising amino acids as set forth in SEQ ID NO:11, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:11; and a second light chain comprising amino acids as set forth in SEQ ID NO:12, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:12; or (iii) a first heavy chain comprising amino acids as set forth in SEQ ID NO: 13, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 13; a second heavy chain comprising amino acids as set forth in SEQ ID NO: 14, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 14; a first light chain comprising amino acids as set forth in SEQ ID NO: 15, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 15; and a second light chain comprising amino acids as set forth in SEQ ID NO: 16, or a sequence of amino acids that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:

16.

26. A nucleic acid comprising a nucleotide sequence encoding the antibody according to any one of claims 1 to 25.

27. A vector comprising the nucleic acid according to claim 26.

28. A host cell comprising a nucleic acid according to claim 26 or a vector according to claim 27.

29. A pharmaceutical composition comprising (i) an antibody according to any one of claims 1-25; and (ii) a pharmaceutically acceptable carrier or excipient.

30. The pharmaceutical composition according to claim 29, further comprising a second therapeutic agent.

31. The pharmaceutical composition according to claim 30, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent and a small molecule drug.

32. A conjugate comprising an antibody according to any one of claims 1 to 25, and a chemical moiety conjugated thereto.

33. The conjugate according to claim 32, wherein the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety and an immunostimulatory molecule.

34. A method of treating a disease in a subject, comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 25, a pharmaceutical composition according to any one of claims 29 to 31 or a conjugate according to claim 32 or 33.

35. The method of claim 34, wherein the disease is cancer or an inflammatory disease.

36. The method of claim 35, wherein the cancer is selected from the group consisting of cervical cancer, lung cancer, liver cancer, breast cancer, and colon cancer.

37. The method of claim 35, wherein the inflammatory disease is selected from rheumatoid arthritis, drug-induced hepatitis, liver fibrosis, chronic obstructive pulmonary disease, asthma, and atopic dermatitis.

38. The method of any one of claims 34-37, further comprising administering to the subject a second therapeutic agent.

39. The method according to claim 38, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

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