An anti-human ifn-alpha5 antibody and conjugates, detection kits and uses thereof

Abstract

The present application relates to the field of biological detection, in particular to an anti-human IFN-alpha 5 antibody and conjugate thereof, a detection kit and application. The present application provides an anti-human IFN-alpha 5 monoclonal antibody, and a kit comprising the antibody or conjugate thereof and a detection method thereof. The kit provided by the present application can complete quantitative detection of human IFN-alpha 5, has high specificity and detection sensitivity, and has good development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of biological detection. Specifically, this invention relates to an anti-human IFN-α5 antibody and its conjugates, a detection kit, and its applications. Background Technology

[0002] Interferons are an important class of cytokines with broad-spectrum antiviral, anti-cell division, and immunomodulatory activities in the same cell type. More than 10 years of clinical application research has shown that they are important antiviral and antitumor therapeutic drugs. To date, it is known that interferons in mammals such as humans, mice, cattle, and horses have three types: α, β, and γ. The α type has many subtypes, which can be divided into two major families. Human interferon family I (human IFN-α1) has 15-20 gene members, most of which encode functional proteins and share approximately 90% homology at the nucleotide level. Human interferon family II (human IFN-α2) has 5-6 members, which share 50% homology with the αI family, and most of these are pseudogenes. Human interferon α5 (human IFN-α5) has low homology with α1 / α2, only about 60-70%. The unique structure of human IFN-α5 can activate different STAT complexes (such as STAT3), resulting in significant differences in gene expression profiles.

[0003] Human IFN-α5, produced by macrophages, not only possesses antiviral activity but also participates in innate immunity. It is one of the genes associated with acute viral bronchiolitis (AVB) caused by respiratory syncytial virus (RSV), determining susceptibility to RSV bronchiolitis. During influenza virus infection, overexpression of IFN-α1 / 2 may lead to uncontrolled inflammatory responses and pathological damage. Human IFN-α5 is primarily expressed in low-pathogenic infectious bronchitis virus (IBV). In some tumor cells, IFN-α5 treatment enhances STAT3 phosphorylation and promotes the expression of anti-apoptotic genes, indicating that the binding of IFN-α5 to the IFNAR receptor favors STAT3 activation rather than STAT1 / STAT2, participating in cell proliferation or apoptosis. This characteristic of IFN-α5 makes it a potential candidate for precision therapy, particularly in overcoming IFN-α2 resistance or reducing toxicity.

[0004] In summary, IFN-α5 plays a crucial role in antiviral, immunomodulatory, and antitumor activity. While its function is redundant with other type I interferons, it also produces specific effects through unique receptor interactions and signaling pathways. Therefore, a highly specific method for detecting human IFN-α5 is needed in this field.

[0005] To improve the specificity and sensitivity of CBA detection methods, the current industry consensus is to develop corresponding CBA detection kits using monoclonal antibodies against human IFN-α5. However, currently available CBA detection kits targeting human IFN-α5 generally have low sensitivity and specificity. Furthermore, the mouse monoclonal antibodies used rely on traditional hybridoma methods for development and production, making the preparation process more complex than that of recombinant monoclonal antibodies and resulting in significant batch-to-batch variability. Therefore, detection kits developed using mouse monoclonal antibodies face challenges such as low sensitivity, narrow linear range, and difficulty in controlling batch-to-batch variability.

[0006] Therefore, there is a need in this field to establish a highly sensitive, wide detection range, and highly specific method for detecting human IFN-α5. Summary of the Invention

[0007] The purpose of this invention is to provide a highly sensitive, wide detection range, and highly specific method for detecting interferon α5.

[0008] In response, the present invention provides an anti-human IFN-α5 antibody and an antibody combination, as well as a detection kit comprising the anti-human IFN-α5 antibody and the antibody combination.

[0009] In a first aspect of the invention, an antibody against human IFN-α5 protein is provided, the antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (HCVR), and the light chain comprising a light chain variable region (LCVR), wherein the heavy chain variable region comprises a complementarity-determining region (CDR): The amino acid sequence is CDR-H1 as shown in SEQ ID NO:1 or SEQ ID NO:2; The amino acid sequence is CDR-H2 as shown in SEQ ID NO:3 or SEQ ID NO:4; The amino acid sequence is CDR-H3 as shown in SEQ ID NO:5 or SEQ ID NO:6; The light chain variable region includes the following complementary determinant regions (CDRs): The amino acid sequence is CDR-L1 as shown in SEQ ID NO:7 or SEQ ID NO:8; The amino acid sequence is CDR-L2 as shown in KAS or YAS. The amino acid sequence is CDR-L3 as shown in SEQ ID NO:9 or SEQ ID NO:10.

[0010] In another preferred embodiment, the antibody has the following CDR: The amino acid sequence is CDR-H1 as shown in SEQ ID NO:1. The amino acid sequence is CDR-H2 as shown in SEQ ID NO:3. The amino acid sequence is CDR-H3 as shown in SEQ ID NO:5. The amino acid sequence is CDR-L1 as shown in SEQ ID NO:7. The amino acid sequence is CDR-L2 as shown in KAS. The amino acid sequence is CDR-L3 as shown in SEQ ID NO:9; or, The amino acid sequence is CDR-H1 as shown in SEQ ID NO:2. The amino acid sequence is CDR-H2 as shown in SEQ ID NO:4. The amino acid sequence is CDR-H3 as shown in SEQ ID NO:6. The amino acid sequence is CDR-L1 as shown in SEQ ID NO:8. The amino acid sequence is CDR-L2 as shown in YAS. The amino acid sequence is CDR-L3 as shown in SEQ ID NO:10.

[0011] In another preferred embodiment, the heavy chain variable region and the light chain variable region further include a skeleton region (FR).

[0012] In another preferred embodiment, the CDR1, CDR2 and CDR3 of the light and heavy chain variable regions are separated by the skeletal regions FR1, FR2, FR3 and FR4, respectively.

[0013] In another preferred embodiment, the HCVR amino acid sequence of the antibody is as shown in SEQ ID NO:11 or SEQ ID NO:12, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0014] In another preferred embodiment, the LCVR amino acid sequence of the antibody is as shown in SEQ ID NO:13 or SEQ ID NO:14, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0015] In another preferred embodiment, the antibody has an HCVR having an amino acid sequence as shown in SEQ ID NO:11, and / or an LCVR having an amino acid sequence as shown in SEQ ID NO:13.

[0016] In another preferred embodiment, the antibody’s HCRV has an amino acid sequence as shown in SEQ ID NO:12, and / or LCVR has an amino acid sequence as shown in SEQ ID NO:14.

[0017] In another preferred embodiment, the light chain and / or heavy chain of the antibody further include a constant region.

[0018] In another preferred embodiment, the constant region is a human-sourced constant region.

[0019] In another preferred embodiment, the heavy chain amino acid sequence of the antibody is as shown in SEQ ID NO:15 or SEQ ID NO:16, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0020] In another preferred embodiment, the light chain amino acid sequence of the antibody is as shown in SEQ ID NO:17 or SEQ ID NO:18, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0021] In another preferred embodiment, the heavy chain of the antibody has an amino acid sequence as shown in SEQ ID NO:15, and / or the light chain has an amino acid sequence as shown in SEQ ID NO:17.

[0022] In another preferred embodiment, the heavy chain of the antibody has an amino acid sequence as shown in SEQ ID NO:16, and / or the light chain has an amino acid sequence as shown in SEQ ID NO:18.

[0023] In another preferred embodiment, any of the above-mentioned amino acid sequences further includes a derived sequence that has optionally been added, deleted, modified and / or substituted at least one amino acid and is capable of retaining the binding affinity of human IFN-α5 protein.

[0024] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 30% of the total number of amino acids in the initial amino acid sequence, preferably 20%, and more preferably 10%.

[0025] In another preferred embodiment, the antibody is an animal-derived antibody, a chimeric antibody, or a humanized antibody.

[0026] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0027] In a second aspect of the invention, a fusion protein is provided, the fusion protein comprising: (1) The antibody as described in the first aspect of the present invention; and (2) Optional tag sequence for expression and / or purification.

[0028] In another preferred embodiment, the label includes an Fc label, a FLAG label, a 6His label, or a combination thereof.

[0029] In a third aspect of the invention, a polynucleotide is provided that encodes an antibody as described in the first aspect of the invention or a fusion protein as described in the second aspect of the invention.

[0030] In a fourth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the third aspect of the invention.

[0031] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector includes viral vectors, such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0032] In a fifth aspect of the invention, a genetically engineered host cell is provided, the host cell containing a vector as described in the fourth aspect of the invention, or having an exogenous polynucleotide as described in the third aspect of the invention integrated into its genome.

[0033] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0034] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0035] In a sixth aspect of the invention, an antibody conjugate is provided, the antibody conjugate comprising: (a) An antibody portion, wherein the antibody portion is an antibody as described in the first aspect of the present invention; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of detectable markers, drugs, or combinations thereof.

[0036] In another preferred embodiment, the antibody portion is coupled to the detectable marker via a chemical bond or a linker.

[0037] In another preferred embodiment, the detectable marker is selected from the group consisting of: fluorescent or luminescent markers, biotin, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, gold nanoparticles / nanorobars, magnetic nanoparticles, or any form of nanoparticles capable of producing a detectable product.

[0038] In another preferred embodiment, the detectable marker is biotin.

[0039] In another preferred embodiment, the drug is a small molecule drug, a biological factor, or a combination thereof.

[0040] In another preferred embodiment, the drug is a cytotoxic drug (toxin).

[0041] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0042] In a seventh aspect of the invention, an antibody ensemble against human IFN-α5 protein is provided, the antibody ensemble comprising antibodies S1 and S2, wherein the heavy chain variable region of antibody S1 includes the following complementarity-determining region (CDR): The amino acid sequence is CDR-H1 as shown in SEQ ID NO:1. The amino acid sequence is CDR-H2 as shown in SEQ ID NO:3. The amino acid sequence is CDR-H3 as shown in SEQ ID NO:5. The light chain variable region of antibody S1 includes the following complementarity-determining regions (CDRs): The amino acid sequence is CDR-L1 as shown in SEQ ID NO:7. The amino acid sequence is CDR-L2 as shown in KAS. The amino acid sequence is CDR-L3 as shown in SEQ ID NO:9; and The heavy chain variable region of antibody S2 includes the following complementarity-determining regions (CDRs): The amino acid sequence is CDR-H1 as shown in SEQ ID NO:2. The amino acid sequence is CDR-H2 as shown in SEQ ID NO:4. The amino acid sequence is CDR-H3 as shown in SEQ ID NO:6; The light chain variable region of antibody S2 includes the following complementarity-determining regions (CDRs): The amino acid sequence is CDR-L1 as shown in SEQ ID NO:8. The amino acid sequence is CDR-L2 as shown in YAS. The amino acid sequence is CDR-L3 as shown in SEQ ID NO:10.

[0043] In another preferred embodiment, the heavy chain variable region and the light chain variable region further include a skeleton region (FR).

[0044] In another preferred embodiment, the CDR1, CDR2 and CDR3 of the light and heavy chain variable regions are separated by the skeletal regions FR1, FR2, FR3 and FR4, respectively.

[0045] In another preferred embodiment, the HCVR of antibody S1 has an amino acid sequence as shown in SEQ ID NO:11 and / or the LCVR has an amino acid sequence as shown in SEQ ID NO:13.

[0046] In another preferred embodiment, the HCRV of antibody S2 has an amino acid sequence as shown in sequence SEQ ID NO:12 and / or the LCVR has an amino acid sequence as shown in SEQ ID NO:14.

[0047] In another preferred embodiment, the light chain and / or heavy chain of the antibody further include a constant region.

[0048] In another preferred embodiment, the constant region is a human-sourced constant region.

[0049] In another preferred embodiment, the heavy chain of antibody S1 has an amino acid sequence as shown in SEQ ID NO:15, and / or the light chain has an amino acid sequence as shown in SEQ ID NO:17.

[0050] In another preferred embodiment, the heavy chain of antibody S2 has an amino acid sequence as shown in SEQ ID NO:16, and / or the light chain has an amino acid sequence as shown in SEQ ID NO:18.

[0051] In another preferred embodiment, any of the above-mentioned amino acid sequences further includes a derived sequence that has optionally been added, deleted, modified and / or substituted at least one amino acid and is capable of retaining the binding affinity of human IFN-α5 protein.

[0052] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 30% of the total number of amino acids in the initial amino acid sequence, preferably 20%, and more preferably 10%.

[0053] In another preferred embodiment, the antibody is an animal-derived antibody, a chimeric antibody, or a humanized antibody.

[0054] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0055] In an eighth aspect of the invention, use is provided for preparing: antibodies as described in the first aspect of the invention, polynucleotides as described in the third aspect of the invention, vectors as described in the fourth aspect of the invention, host cells as described in the fifth aspect of the invention, antibody conjugates as described in the sixth aspect of the invention, or antibody combinations as described in the seventh aspect of the invention. i) Reagents or kits for detecting human IFN-α5 protein; ii) Preparations or drugs for treating human IFN-α5-related diseases.

[0056] In another preferred embodiment, the human IFN-α5-related diseases are selected from the group consisting of: inflammation-related diseases, tumors, autoimmune diseases, viral infections, or combinations thereof.

[0057] In another preferred embodiment, the preparation or drug is a liquid dosage form, preferably an injection.

[0058] In another preferred embodiment, the drug is a cell therapy drug.

[0059] In a ninth aspect of the present invention, a human IFN-α5 protein detection kit is provided, the kit comprising an antibody as described in the first aspect of the present invention, an antibody-drug conjugate as described in the sixth aspect of the present invention, or an antibody combination as described in the seventh aspect of the present invention.

[0060] In another preferred embodiment, the detection kit is used for double-antibody sandwich assay.

[0061] In another preferred embodiment, the detection kit is used for CBA detection, ELISA detection, and / or colloidal gold detection.

[0062] In another preferred embodiment, the kit is a CBA kit.

[0063] In another preferred embodiment, the detection kit comprises an antibody combination as described in the seventh aspect of the invention, wherein antibody S1 is immobilized on a carrier and used to capture human IFN-α5 protein, and antibody S2 is conjugated to a detection marker.

[0064] In another preferred embodiment, the detection kit comprises microspheres (such as fluorescent microspheres) coated with the antibody S1.

[0065] In another preferred embodiment, the test kit includes an antibody S2 containing biotin.

[0066] In another preferred embodiment, the detection kit further includes a human IFN-α5 protein standard.

[0067] In another preferred embodiment, the detection kit further comprises components selected from the group consisting of: fluorescently labeled streptavidin, lyophilized human IFN-α5 standard, washing buffer, sample dilution buffer, matrix buffer, or combinations thereof.

[0068] In another preferred embodiment, the fluorescent protein is phycoerythrin.

[0069] In a tenth aspect of the present invention, a method for detecting human IFN-α5 in a sample is provided, using an antibody as described in the first aspect of the present invention, an antibody conjugate as described in the sixth aspect of the present invention, an antibody combination as described in the seventh aspect of the present invention, or a detection kit as described in the ninth aspect of the present invention.

[0070] In another preferred embodiment, the method includes the following steps: 1) Immobilize antibody S1 onto the vector; 2) Label antibody S2 using a detectable marker; 3) The antibodies S1 and S2 and the sample to be tested are incubated together, so that the antibodies S1 and S2 bind to human IFN-α5 in the sample; 4) Wash away the reagents that are not bound to the carrier, and detect the detectable marker to detect human IFN-α5.

[0071] In another preferred embodiment, the detection is a quantitative detection.

[0072] In another preferred embodiment, the carrier is a microsphere, preferably a fluorescent microsphere.

[0073] In another preferred embodiment, the detectable marker is biotin.

[0074] In another preferred embodiment, step 4) includes the following steps: Avidin labeled with fluorescent protein is added and incubated to link biotin to antibody S2, and the fluorescent protein is then detected.

[0075] In another preferred embodiment, the avidin is streptavidin.

[0076] In another preferred embodiment, the fluorescent protein is phycoerythrin.

[0077] In another preferred embodiment, the method further includes the steps of: detecting human IFN-α5 standards to obtain a standard curve, and calculating the content of human IFN-α5 in the sample to be tested based on the standard curve.

[0078] In another preferred embodiment, the sample to be tested is selected from the group consisting of cell culture medium, whole blood, serum, plasma, body fluid, or combinations thereof.

[0079] In another preferred embodiment, the method is an in vitro method.

[0080] In another preferred embodiment, the method is a non-therapeutic and non-diagnostic method.

[0081] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0082] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0083] Figure 1 The results of immunizing mice with human IFN-α5 are shown. The horizontal axis represents the tail vein serum dilution factor, and the vertical axis represents the OD450 value. OD450 is the absorbance at 450 nm.

[0084] Figure 2 The binding results of anti-human IFN-α5 monoclonal antibodies 1# and 2# to human IFN-α1, IFN-α2, IFN-α5, IFN-β, and IFN-γ are shown. The ordinate represents the absorbance at 450 nm, and the abscissa represents the interferon type.

[0085] Figure 3 The standard curve for human IFN-α5 concentration detection is shown. The horizontal axis represents the log10 value of the calibrator concentration, and the vertical axis represents the log10 value of the fluorescence value. MFI represents the average fluorescence intensity. Detailed Implementation

[0086] Through extensive and in-depth research, the inventors have developed, for the first time, a highly specific anti-human IFN-α5 antibody. Specifically, this invention provides an anti-human IFN-α5 monoclonal antibody and a human IFN-α5 CBA detection kit containing this antibody. The kit of this invention exhibits higher sensitivity and a wider detection range. It also possesses extremely high specificity, avoiding interference from human IFN-α1, IFN-α2, IFN-β, and IFN-γ, resulting in more accurate detection and demonstrating promising development and application prospects. Based on this, this invention was completed.

[0087] the term To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0088] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0089] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0090] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0091] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0092] Interferon α5 In this specification, the term "interferon α5 (IFN-α5)" refers to a cytokine belonging to the type I interferon family. Human IFN-α5 is primarily produced by macrophages and leukocytes, and has a molecular weight of approximately 19 kDa. The amino acid sequence of human IFN-α5 is shown in SEQ ID NO:19.

[0093] SEQ ID NO:19: MALPFVLLMALVVLNCKSICSLGCDLPQTHSLSNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDLEACMMQEVGVEDTPLMNVDSILTVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSANLQERLRRKE Human IFN-α5, produced by macrophages, not only possesses antiviral activity but also participates in innate immunity and can influence tumor cell proliferation or apoptosis. This characteristic of IFN-α5 makes it a potential candidate for precision therapy, particularly in overcoming IFN-α2 resistance or reducing toxicity.

[0094] As a member of the type I interferon family, IFN-α5 protein possesses diverse biological functions, primarily involving immune regulation, antiviral defense, and antitumor activity. Its subtype-specific functions result in different gene expression profiles, thus highlighting its antiviral or antitumor effects.

[0095] Antiviral effects: IFN-α5 directly inhibits viral replication by inducing the expression of interferon-stimulated genes (ISGs), such as protein kinase R (PKR), 2'-5' oligoadenylate synthase (OAS), and MX GTPase. IFN-α5 can also enhance the activity of natural killer (NK) cells, dendritic cells, and macrophages, promoting viral clearance.

[0096] Immunomodulatory functions: IFN-α5 promotes the expression of major histocompatibility complex (MHC) molecules, enhancing antigen presentation and increasing its efficiency. Secondly, IFN-α5 can also regulate the Th1 / Th2 response, favoring Th1-type immune responses and enhancing cellular immunity. At appropriate levels, it coordinates inflammatory responses; overexpression may be involved in autoimmune diseases.

[0097] Antitumor activity: First, IFN-α5 inhibits tumor cell growth by inducing cell cycle arrest or apoptosis. Second, it can activate NK cells and cytotoxic T lymphocytes, promoting tumor cell recognition and clearance. Third, it can downregulate pro-angiogenic factors (such as VEGF), limiting tumor blood supply.

[0098] Subtype-specific functions: IFN-α5 may exhibit differences in downstream signal intensity or duration due to varying affinity for the IFNAR receptor. Because IFN-α5 possesses diverse gene expression profiles, it can activate unique STAT complex combinations (such as STAT3), influencing the expression of specific genes and thus demonstrating significant antiviral or antitumor effects.

[0099] Clinical Applications and Research: Treatment of viral infections: It has potential applications in chronic hepatitis B, hepatitis C, etc., but the commonly used subtype in clinical practice is IFN-α2, and research on IFN-α5 is mostly in the experimental stage.

[0100] Cancer treatment exploration: It has shown anti-proliferative effects in models of melanoma, leukemia, etc., and may be used in combination with chemotherapy or immune checkpoint inhibitors.

[0101] Autoimmune risks: Attention should be paid to the potential side effects of inducing autoimmune responses, and the risks may vary depending on the subtype.

[0102] In recent years, an increasing number of studies have shown that IFN-α5 has positive effects in the treatment of viral infections, chronic hepatitis B, hepatitis C, and in the field of anti-tumor therapy. However, the commonly used subtype in clinical practice is IFN-α2, and research on IFN-α5 is mostly in the experimental stage.

[0103] In conclusion, the level of IFN-α5 protein is of great significance for the diagnosis and treatment of viral infections, the assessment of immune status, the monitoring of tumor treatment, and medical research.

[0104] This invention provides antibodies, detection kits, and detection methods for detecting IFN-α5 (especially human IFN-α5).

[0105] Antibody In this application, the term "antibody" should be interpreted in the broadest sense, encompassing various antibody structures, including but not limited to Y-type antibodies, so-called full-length antibodies, antigen-binding portions of Y-type antibodies, and their genetic or chemical modifications. The "antigen-binding portion" refers to one or more portions or fragments of a Y-type antibody that retain the antibody's ability to specifically bind to human IFN-α5.

[0106] In this application, the term "monoclonal antibody" (mAb) refers to a highly homogeneous group of antibodies having substantially the same antigenic determinants. That is, within this antibody group, individual antibodies are substantially identical, except for a small number of mutations that may occur naturally. A monoclonal antibody can exhibit a single binding specificity and affinity for a specific epitope on an antigen. In contrast to polyclonal antibodies, which typically contain antibodies targeting different epitopes, each monoclonal antibody can target the same or substantially the same epitope on an antigen. The modifier "monoclonal" indicates that the antibody's properties are derived from a substantially homogeneous group of antibodies and should not be interpreted as an antibody requiring preparation by any particular method. Such antibodies can be prepared by a variety of methods, including, but not limited to, hybridoma methods, recombinant DNA methods, phage antibody libraries, and similar methods.

[0107] In this application, the term "mouse antibody" or "anti-human IFN-α5 mouse monoclonal antibody" or similar terms, with the modifier "mouse," indicates that the complementarity-determining region (CDR) of the antibody is derived from a murine immunoglobulin sequence. In one embodiment, the anti-human IFN-α5 mouse monoclonal antibody may comprise the CDR and backbone region (FR) of an antibody derived from a murine immunoglobulin sequence. In one embodiment, the anti-human IFN-α5 mouse monoclonal antibody may be an antibody whose CDR region is derived from a murine immunoglobulin sequence, while the FR is derived from a germline immunoglobulin sequence of another mammal (such as rabbit or human). The term "anti-human IFN-α5 mouse monoclonal antibody" may also comprise antibodies having amino acid residues encoded by non-murine immunoglobulin sequences, for example, mutations introduced by in vitro random or point-specific mutations, or mutations introduced by in vivo somatic mutations. However, the term "anti-human IFN-α5 mouse monoclonal antibody" does not include antibodies whose CDR region is derived from a germline of another mammal (such as human).

[0108] In this application, the term "antibody" refers to an immunoglobulin molecule composed of four heterologous polypeptide chains, wherein the two chains with larger molecular weights are called heavy chains (H), and the two chains with smaller molecular weights are called light chains (L). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The approximately 110 amino acid sequences near the N-terminus of both the heavy and light chains exhibit significant variation, while the remaining amino acid sequences are relatively constant. Therefore, the regions of the light and heavy chains with significantly varied amino acid sequences near the N-terminus are called variable regions (V), accounting for 1 / 4 and 1 / 2 of the heavy and light chains, respectively; the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C), accounting for 3 / 4 and 1 / 2 of the heavy and light chains, respectively.

[0109] The V regions of the heavy and light chains are called VH and VL, respectively. Each VH and VL contains three regions with highly variable amino acid composition and sequence, called hypervariable regions (HVRs) or complementarity-determining regions (CDRs), including HVR1 (CDR1), HVR2 (CDR2), and HVR3 (CDR3), with HVR3 (CDR3) exhibiting the highest degree of variation. These three CDRs of VH and VL collectively form the antigen-binding site of the antibody, determining its specificity and serving as the site for antibody recognition and binding to antigens. Within the V region, the amino acid composition and sequence of the regions outside the CDRs are relatively conserved and are called framework regions (FRs). Each VH or VL has four framework regions, denoted as FR1, FR2, FR3, and FR4, respectively.

[0110] The C regions of the heavy chain and light chain are called CH and CL, respectively. The CL lengths of different types (κ or λ) Ig are basically the same, but the CH lengths of different classes of Ig are different. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4.

[0111] In this application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of that interaction. In this application, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0112] In this application, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may also contain a replication initiation site.

[0113] In this application, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. Conservative amino acid substitutions include substitutions that replace amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). These conservative substitutions are preferably produced by amino acid substitutions according to Table 1 below.

[0114] Table 1 Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0115] As used herein, the terms "vector," "expression system," or "expression vector" refer to a nucleic acid sequence containing a desired coding and control sequence operatively linked together, such that a host transformed with these sequences can produce the encoded protein. For transformation, the expression system may be contained on a vector; however, the associated nucleic acid molecules may subsequently be integrated into the host chromosome as well.

[0116] As used herein, the term "host cell" is a cell that supports the replication or expression of an expression vector. Host cells can be prokaryotic cells such as Escherichia coli, or eukaryotic cells such as yeast cells, insect cells, amphibian cells, or mammalian cells.

[0117] As used herein, the terms “transfection,” “stable transfection,” or “transient transfection” refer to the uptake of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed. Various transfection methods are known to those skilled in the art. For example, transfection can be performed in the presence of an expression vector and a high concentration of calcium phosphate, by electroporation, by inserting a phage or viral expression vector into a host cell, by mechanical insertion of nucleic acids, or even by culturing host cells in the presence of unpackaged nucleic acid fragments. Successful transfection is typically confirmed when any indication of manipulation of the vector of interest appears in the host cell.

[0118] Anti-human IFN-α5 monoclonal antibody This application provides an isolated anti-human IFN-α5 monoclonal antibody, wherein the monoclonal antibody comprises three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: The amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 or SEQ ID NO:2; The amino acid sequence of CDR-H2 is shown in SEQ ID NO:3 or SEQ ID NO:4; The amino acid sequence of CDR-H3 is shown in SEQ ID NO:5 or SEQ ID NO:6; The amino acid sequence of CDR-L1 is shown in SEQ ID NO:7 or SEQ ID NO:8; The amino acid sequence of CDR-L2 is shown in KAS or YAS. The amino acid sequence of CDR-L3 is shown in SEQ ID NO:9 or SEQ ID NO:10.

[0119] In one specific embodiment, the CDR-H1 of the antibody provided in this application has the amino acid sequence shown in SEQ ID NO:1; CDR-H2 has the amino acid sequence shown in SEQ ID NO:3. CDR-H3 has the amino acid sequence shown in SEQ ID NO:5. CDR-L1 has the amino acid sequence shown in SEQ ID NO:7. CDR-L2 has the amino acid sequence shown in KAS. CDR-L3 has the amino acid sequence shown in SEQ ID NO:9.

[0120] In one specific embodiment, the CDR-H1 of the monoclonal antibody provided in this application has the amino acid sequence shown in SEQ ID NO:2. CDR-H2 has the amino acid sequence shown in SEQ ID NO:4. CDR-H3 has the amino acid sequence shown in SEQ ID NO:6. CDR-L1 has the amino acid sequence shown in SEQ ID NO:8. CDR-L2 has the amino acid sequence shown in YAS. CDR-L3 has the amino acid sequence shown in SEQ ID NO:10.

[0121] In one specific embodiment, the above-mentioned monoclonal antibody comprises an antibody heavy chain variable region (HCVR) and an antibody light chain variable region (LCVR), wherein: The amino acid sequence of HCVR is shown in SEQ ID NO:11 or SEQ ID NO:12; The amino acid sequence of LCVR is shown in SEQ ID NO:13 or SEQ ID NO:14.

[0122] In one specific embodiment, the monoclonal antibody provided in this application has an HCVR amino acid sequence as shown in SEQ ID NO:11 and an LCVR amino acid sequence as shown in SEQ ID NO:13.

[0123] In one specific embodiment, the HCRV of the monoclonal antibody provided in this application has the amino acid sequence shown in SEQ ID NO:12, and the LCVR has the amino acid sequence shown in SEQ ID NO:14.

[0124] In one specific embodiment, the above-mentioned monoclonal antibody comprises a heavy chain and a light chain, wherein: The amino acid sequence of the heavy chain is shown in SEQ ID NO:15 or SEQ ID NO:16; The amino acid sequence of the light chain is shown in SEQ ID NO:17 or SEQ ID NO:18.

[0125] In one specific embodiment, the anti-human IFN-α5 monoclonal antibody of this application is anti-human IFN-α5 monoclonal antibody 1#, whose heavy chain has the amino acid sequence shown in SEQ ID NO:15 and whose light chain has the amino acid sequence shown in SEQ ID NO:17.

[0126] In one specific embodiment, the anti-human IFN-α5 monoclonal antibody of this application is anti-human IFN-α5 monoclonal antibody 2#, whose heavy chain has the amino acid sequence shown in SEQ ID NO:16 and whose light chain has the amino acid sequence shown in SEQ ID NO:18.

[0127] In this specification, an “isolated” antibody is an antibody that has been separated from components of its native environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). Methods for evaluating antibody purity are well known in the art, and may be found, for example, in Flatman et al., J. Chromatogr. B848: 79-87 (2007).

[0128] In this specification, "monoclonal antibody" means an antibody derived from a substantially homologous group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, and such variants are typically present in trace amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles). Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0129] In this specification, "anti-human IFN-α5 monoclonal antibody" means a monoclonal antibody that can bind to human IFN-α5 with sufficient affinity, such that the monoclonal antibody can be used as a disease diagnostic agent and / or therapeutic agent targeting human IFN-α5.

[0130] In this specification, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD). Affinity can be measured using methods commonly known in the art.

[0131] The anti-human IFN-α5 monoclonal antibody of this application does not bind to proteins unrelated to the target. Here, "unrelated protein" refers to proteins other than human IFN-α5, which is the target; and "does not bind" means that, when the binding ability of the anti-human IFN-α5 monoclonal antibody of this invention to human IFN-α5, which is its target, is taken as 100%, the binding ability of the anti-human IFN-α5 monoclonal antibody of this application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0132] The anti-human IFN-α5 monoclonal antibody of this application does not bind to interferon α5 from other animal species. Here, "other animal species" refers to animal species other than humans, such as rhesus monkeys, cynomolgus monkeys, rats, mice, etc.; here, "not binding" means that when the binding ability of the anti-human IFN-α5 monoclonal antibody of this invention to human IFN-α5 as its target is taken as 100%, the binding ability of the anti-human IFN-α5 monoclonal antibody of this invention to IFN-α5 from other animal species is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0133] The anti-human IFN-α5 monoclonal antibody of this application has an equilibrium dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤50 nM, or ≤40 nM for human IFN-α5.

[0134] Furthermore, all anti-human IFN-α5 monoclonal antibodies in this application are mouse monoclonal antibodies.

[0135] CBA Cytometric Bead Array (CBA) is a multiplex protein quantification method based on flow cytometry. It can simultaneously detect multiple indicators in a single sample. BD Biosciences utilizes the amplification of fluorescence signals by flow cytometry to attach soluble analytes to microparticles with a diameter similar to that of cells, allowing for the detection of various soluble factors in the sample. The basic principle of CBA is similar to ELISA: tiny, dispersed particles capture liquid analytes, and flow cytometry detects the fluorescence emitted by the "sandwich"-like particle-analyte complex, thus determining the quantity of the analyte.

[0136] Each CBA microsphere is uniform in size and has a specific fluorescence intensity. It is coated with a specific capture antibody suitable for a particular analysis (such as other antibodies or soluble proteins), providing a capture surface similar to an ELISA plate. When the microspheres are mixed with the sample solution, the specific antibody on the microsphere binds to the corresponding antigen or protein in the sample (serum, plasma, or cell culture medium). Then, a fluorescently labeled detection antibody is added, forming a "sandwich" complex. Finally, the specific target protein is detected by flow cytometry. Each CBA microsphere carries a different intensity of red fluorescence; the target protein is qualitatively identified by detecting the difference in fluorescence intensity of the microspheres in the FL3 channel of the flow cytometer. The detection antibody is labeled with PE fluorescein and is detected in the FL2 channel of the flow cytometer; the target protein is quantified by detecting the PE fluorescence intensity of the antibody.

[0137] In this specification, "avidin" is a glycoprotein, each molecule of which consists of four subunits and can bind tightly to four biotin molecules. Streptavidin, extracted from Streptomyces, is more commonly used.

[0138] In this specification, biotin is a small molecule growth factor widely distributed in plants and animals, also known as coenzyme R or vitamin H. Avidin is a basic glycoprotein extracted from ovalbumin, which has a very high affinity for biotin. The reaction between the enzyme-bound avidin molecule and the biotin molecule bound to a specific antibody amplifies the reaction, and the color development due to the enzyme's catalytic action upon encountering the corresponding substrate allows for the determination of the amount of antibody bound. Therefore, combining avidin and biotin with CBA can significantly improve the sensitivity of CBA. The biotin-avidin system has various applications in CBA, including indirect coating and final reaction amplification. The enzyme-labeled antibody in conventional CBA can also be replaced with a biotinylated antibody, which is then linked to an avidin-enzyme conjugate to amplify the reaction signal.

[0139] Test kit This invention provides a detection kit for detecting human IFN-α5, which contains the antibody or antibody combination of this invention.

[0140] In one embodiment, the test kit uses a double-antibody sandwich method for detection. The detection method of the present invention can be used with CBA detection.

[0141] In one specific embodiment, the anti-human interleukin monoclonal antibody in the kit of this application comprises a first antibody and a second antibody, wherein the first antibody is a coating antibody that has been immobilized on a solid-phase support (such as microspheres); the second antibody is a detection antibody that is labeled with biotin. Preferably, the microspheres are fluorescent microspheres. More preferably, the particle size is 5 μm. In a specific embodiment, polystyrene microspheres from China Nanomicro can be used.

[0142] In one specific embodiment, the first antibody and the second antibody are the anti-human IFN-α5 monoclonal antibodies of this application.

[0143] In one specific embodiment, the first antibody in the CBA kit is anti-human IFN-α5 monoclonal antibody 1#, whose heavy chain has the amino acid sequence shown in SEQ ID NO:15 and whose light chain has the amino acid sequence shown in SEQ ID NO:17; the second antibody is anti-human IFN-α5 monoclonal antibody 2#, whose heavy chain has the amino acid sequence shown in SEQ ID NO:16 and whose light chain has the amino acid sequence shown in SEQ ID NO:18.

[0144] Furthermore, the CBA kit also includes the apparatus or reagents necessary for the detection of IFN-α5.

[0145] Specifically, the CBA kit also includes streptavidin labeled with peroxidase or fluorescent protein, IFN-α5 standard, substrate, coating antibody diluent, washing buffer, blocking / sample diluent, and stop solution.

[0146] In one specific embodiment, the fluorescent protein is preferably phycoerythrin (PE), and the streptavidin labeled thereon is a streptavidin-phycoerythrin conjugate (SA-PE).

[0147] In one specific embodiment, the IFN-α5 standard in the kit is human IFN-α5 protein; the coating antibody diluent is phosphate-buffered saline (PBS) pH 7.4; the washing buffer is phosphate-buffered saline (PBS) containing 0.05% Tween 20; and the blocking / sample diluent is phosphate-buffered saline (PBS) containing 0.5% BSA, 0.05% Tween 20, and 0.05% Proclin 300.

[0148] Preferably, the CBA kit comprises the following components: 1) Pre-coated antibody microspheres: Anti-human IFN-α5 monoclonal antibody 1# (i.e., primary antibody), 2.5 mL / tube, 1 tube; 2) Antibody detection: Anti-human IFN-α5 monoclonal antibody 2# (i.e., the second antibody) (labeled with biotin), 2.5 mL / tube, 1 tube; 3) Standard: Lyophilized human IFN-α5 protein, 10 ng / tube, 1 tube; 4) Streptavidin-phycoerythrin conjugate (SA-PE), 2.5 mL / tube, 1 tube; 5) Matrix buffer: Contains BSA and protein protectant, 2.5 ml / tube, 1 tube; 6) Washing buffer: Phosphate buffer (10XPBST), pH 7.4, 5ml / bottle, 1 bottle; 7) Sample dilution solution: 0.5% BSA in PBS, pH 7.4, 5ml / bottle, 1 bottle.

[0149] Detection methods This invention also provides a method for detecting IFN-α5 using the antibodies or kits of this invention. The detection method of this invention can be a double-antibody sandwich assay. The detection method of this invention can use CBA detection.

[0150] This invention uses biotin labeling with anti-human IFN-α5 monoclonal antibody, which can form an antibody-biotin-avidin system (ABAS) with streptavidin labeled with phycoerythrin. The strong binding with high affinity can have a multi-stage amplification effect, giving the kit of this application higher sensitivity and a wider detection range.

[0151] One specific method involves binding a known amount of a first antibody to the surface of a solid-phase carrier. The sample to be tested is then applied to the surface, allowing any IFN-α5 material present in the cells and / or serum to be captured by the immobilized first antibody. Unbound material is preferably removed by one or more washing steps. A second antibody, labeled with biotin and serving as the detection antibody, is then added, allowing binding of any IFN-α5 material captured by the first antibody in the cells and / or serum, such that each unit of IFN-α5 simultaneously binds to both antibodies, forming a "sandwich". The amount of the bound second antibody is then determined in a direct or indirect detection method. Specifically, the label or enzyme can be directly or indirectly linked to the second antibody via a link such as a biotin-streptavidin or biotin-avidin linker.

[0152] In one embodiment, the detection method of the present invention includes the following steps: Fluorescent microspheres were coated with anti-human IFN-α5 monoclonal antibody 1# (i.e., the first antibody); Biotin was labeled onto anti-human IFN-α5 monoclonal antibody 2# (i.e., the second antibody); Microspheres coated with the first antibody, the test sample, and the biotin-labeled second antibody were sequentially added to a flow cytometer and incubated. Phycoerythrin-labeled streptavidin was added to a flow cytometer and incubated. A standard curve was fitted using the MFI value of human IFN-α5 standard, and the content of human IFN-α5 in the test sample was calculated by substituting the MFI value of the test sample into the equation.

[0153] In one specific embodiment, the method for quantitatively detecting the human IFN-α5 content in a sample using the CBA kit of this application includes the following steps: (1) Coating: The anti-human IFN-α5 monoclonal antibody 1# (i.e., the first antibody) was prepared into a working solution with a concentration of 1 g / L using the coating antibody dilution buffer (phosphate buffer (PBS, pH 7.4). Then, it was mixed in a clean EP at a ratio of 10 μg / 1E7 microspheres and incubated at room temperature for 2 hours. (2) Blocking: Centrifuge to discard the coated antibody working solution in the EP tube, wash twice with washing buffer (phosphate buffer (PBS) containing 0.05% Tween 20), then add blocking buffer (phosphate buffer (PBS) containing 0.5% BSA, 0.05% Tween 20 and 0.05% Proclin 300) at a volume of 100 μL / well, and place on a shaker (120 rpm) at room temperature for 2 hours; (3) Preparation of protein standards: Take 7 EP tubes and number them sequentially. Add 150 μL of sample diluent to each of tubes 1-6 and place them on the EP tube rack. Prepare a 10 μg / L concentration of standard protein (human IFN-α5 protein) using the sample diluent. Add 50 μL to the first EP tube, then add 50 μL of the liquid from the first EP tube to the second EP tube for a fourfold dilution, and so on up to the sixth EP tube. The concentrations are 10000 pg / mL, 2500 pg / mL, 625 pg / mL, 156.3 pg / mL, 39.6 pg / mL, 9.7 pg / mL, and 2.4 pg / mL, respectively. (4) Sample loading: Add microspheres coated with the first antibody, protein standard solution, and biotin-labeled second antibody in sequence according to the dosage of 25 μL / tube, and incubate at room temperature for 2 hours on a shaker (500 rpm); (5) Add SA-PE: Add 25 μL / tube to EP and incubate on a shaker (500 rpm) for 0.5 hours; (6) Washing: Centrifuge to discard the reaction solution in the EP tube, wash twice with washing solution, and resuspend the microspheres with sample diluent; (7) Detection: The MFI value of each EP tube was measured at a wavelength of 585nm using a flow cytometer, and a standard curve was fitted based on the MFI value of the standard.

[0154] The antibody, detection kit, and detection method of this application can be used to detect human IFN-α5 in cell culture medium or human serum.

[0155] Pharmaceutical Composition The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0156] The pharmaceutical compositions of the present invention may contain any antitumor drug (such as an antitumor antibody) linked to the anti-human IFN-α5 antibody of the present invention, and thus may be used to treat tumors. Furthermore, other therapeutic agents may be used simultaneously.

[0157] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described nanobody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0158] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0159] The main advantages of this invention include: 1) This invention provides antibodies and antibody pairs (antibody combinations) that specifically target human IFN-α5. When used in a CBA kit to detect human IFN-α5, these antibodies significantly expand the detection range and improve detection sensitivity. Repeated assays demonstrate high accuracy.

[0160] 2) The antibodies and combinations of the present invention do not cross-react with human IFN-α1, human IFN-α2, human IFN-β, and human IFN-γ, thus avoiding interference from similar cytokines and improving detection specificity.

[0161] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0162] Example 1: Screening and preparation of anti-human IFN-α5 monoclonal antibodies Human IFN-α5 (amino acid sequence shown in SEQ ID NO:19) was prepared and used to immunize Balb / c mice. Post-immunization, serum from the tail vein of the mice was collected to determine the immunogenicity. After the immunogenicity reached the target level, venous blood from the mice was collected to prepare a B cell suspension for sorting. Figure 1 The results of immunization of mice with human IFN-α5 are shown. The horizontal axis represents the dilution factor after collecting tail vein serum from different mice, and the vertical axis represents the OD450nm value. The results show that the immunogenicity reached the expected level. One mouse was selected for single B cell sorting. First, specific memory B cells were screened using cell surface markers and antigens. Then, the DeepLight On-chip Cell Screening platform was used for sorting to obtain specific plasma cells. Single B cells that bind to human IFN-α5 were selected. After a short period of culture, Binding ELISA was used for forward / reverse screening to select monoclonal antibodies that bind to human IFN-α5 but have no binding activity with human IFN-α1, human IFN-α2, human IFN-β, and human IFN-γ. These monoclonal antibodies (antibody 1# and antibody 2#) were obtained. The obtained first and second antibodies are mouse monoclonal antibodies. Human-mouse antibody chimerism was performed on the obtained mouse monoclonal antibodies to obtain human-mouse chimeric antibodies with a mouse variable region and a human constant region. The above immunization and screening processes were outsourced to Detai Biotechnology.

[0163] The heavy and light chain variable region sequences of the first and second antibodies were obtained by sequencing, and their CDR sequences were defined according to Kabat rules. The antibody sequences are shown in Table 2.

[0164] Table 2 Antibody Sequences Example 2: Specificity identification of anti-human IFN-α5 monoclonal antibody 1. Antibody recognition antigen specificity experiment: Human IFN-α1, IFN-α2, IFN-α5, IFN-β, and IFN-γ were diluted to 10 μg / mL with coating buffer (0.05 M, pH 9.6 carbonate buffer) and coated onto an ELISA plate overnight at 2-8°C. The plate was then blocked at room temperature for 2 hours the following day. After washing, 100 μL of diluted cell culture supernatant (culture supernatant of single B cells bound to human IFN-α5) was added. A positive control (serum from immunized mice containing anti-IFN-α5 polyclonal antibody) and a negative control (serum from unimmunized mice) were also included. The plates were incubated at room temperature for 1 hour. After washing, diluted ELISA-labeled secondary antibody was added and incubated at room temperature for 1 hour. Finally, after washing, the plates were developed, and the absorbance at OD450 nm was read. Results are as follows: Figure 2As shown, antibodies #1 and #2 did not bind to human IFN-α1, human IFN-α2, human IFN-β, and human IFN-γ antigens, indicating that antibodies #1 and #2 have good specificity for IFN-α5 protein. These antibodies can be used for pairing experiments. The detection results for antibodies #1 and #2 are shown in Table 3.

[0165] Table 3 Antibody Specificity Detection 2. Anti-human IFN-α5 antibody pairing experiment Two antibodies were biotin-labeled using the Sulfo-NHS-LC-Biotin kit, strictly following the kit instructions. After labeling, the free biotin was removed by dialysis using a dialysis bag; the resulting solution was the biotin-labeled antibody.

[0166] Dilute the two antibody strains to 10 μg / mL with coating buffer, coat fluorescent microspheres at 2-8℃, and incubate at room temperature for 2 hours. After centrifugation, add blocking buffer and block at room temperature for 2 hours. After washing, add storage buffer and count for later use.

[0167] Matrix buffer, human IFN-α5 protein, antibody-coated microspheres, and biotin-labeled antibody were added sequentially, and the mixture was incubated at room temperature for 2 hours. Then, diluted phycoerythrin-labeled streptavidin was added, and the mixture was incubated at room temperature for 0.5 hours. Finally, after washing, the fluorescence values ​​were read using flow cytometry. The results are shown in Table 4 below. Antibodies 1# and 2# can be paired for assembling a quantitative detection kit for human IFN-α5 CBA.

[0168] Table 4 Antibody pairing test results Example 3: Composition of the Human IFN-α5 CBA Quantitative Detection Kit A CBA kit was prepared by coating fluorescent microspheres with antibody 1# as a capture antibody and biotin-labeling antibody 2# as a detection antibody. The reagent composition, specifications, source and storage conditions of the kit are shown in Table 5.

[0169] 1) Pre-coated antibody microspheres: Anti-human IFN-α5 monoclonal antibody 1# (i.e., primary antibody), 2.5 mL / tube, 1 tube; 2) Antibody detection: Anti-human IFN-α5 monoclonal antibody 2# (i.e., the second antibody) (labeled with biotin), 2.5 mL / tube, 1 tube; 3) Standard (or calibrator): Lyophilized human IFN-α5 protein, 10 ng / tube, 1 tube; 4) Streptavidin-phycoerythrin conjugate (SA-PE), 2.5 mL / tube, 1 tube; 5) Matrix buffer: BSA, protein protectant, 5 mL / tube, 1 tube; 6) Washing buffer: Phosphate buffer (10XPBST), pH 7.4, 5 mL / bottle, 1 bottle; The components are shown in Table 5 below: Table 5. Composition of CBA Reagent Kit Example 4: Performance Evaluation of Human IFN-α5 CBA Reagent Kit The human IFN-α5 CBA kit described in Example 3 was used to quantitatively detect human IFN-α5 in the quality control samples.

[0170] 1. Linear range: (1) Prepare protein standards by diluting human IFN-α5 protein with sample diluent (PBS with 0.5% BSA, pH 7.4), and prepare quality control samples by diluting human IFN-α5 protein with cell culture medium as test samples. Fit a standard curve based on the MFI value of the protein standards. Substitute the MFI value of the test samples into the standard curve to obtain the theoretical concentration of human IFN-α5 in the test samples, and then calculate the recovery rate of the test samples (recovery rate = theoretical concentration value / actual concentration value * 100%), thereby analyzing the quantitative detection effect of the CBA kit in Example 3 on human IFN-α5 in cell culture medium.

[0171] (2) Quantitative detection procedure: 1) Preparation of protein standards: Take 8 EP tubes and number them sequentially. Starting from the second tube, add 300 μL of sample diluent to each tube and place them on the EP tube rack. Prepare the protein standard to a concentration of 10 ng / mL in the first tube using the sample diluent. Add 100 μL to the second EP tube for a four-fold dilution, and so on up to the seventh EP tube. The concentrations are 10000 pg / mL, 2500 pg / mL, 625 pg / mL, 156.3 pg / mL, 39.1 pg / mL, 9.8 pg / mL, and 2.4 pg / mL, respectively. 2) Preparation of quality control samples: Take 6 EP tubes and take samples from 10000 pg / mL to prepare quality control samples of 6250 pg / mL, 1250 pg / mL, 250 pg / mL, 50 pg / mL, 10 pg / mL and 2 pg / mL respectively. 3) Sample loading: Add matrix buffer, protein standard solution, microspheres coated with the first antibody, and biotin-labeled second antibody in sequence according to the volume of 25 μL / tube, and incubate on a shaker (500 rpm) at room temperature for 2 hours; 4) Add SA-PE: Add 25 μL / tube to EP and incubate on a shaker (500 rpm) for 0.5 hours; 5) Washing: Centrifuge to discard the reaction solution in the EP tube, wash twice with washing solution, and resuspend the microspheres with sample diluent; 6) Detection: The MFI value of each EP tube was measured by flow cytometer at a wavelength of 585nm. A standard curve was fitted based on the MFI value of the standard, and the MFI value of the quality control sample was substituted into the equation to calculate the concentration of the quality control sample.

[0172] Results and Discussion The linear range detection data of human IFN-α5 standard are shown in Table 6: Table 6. Linear range detection data of human IFN-α5 standard. The detection curve for human IFN-α5 showed satisfactory recovery within the concentration range of 2.4–10,000 pg / mL.

[0173] Curve equations are as follows Figure 3 As shown: y = -0.0004x 4 - 0.0338x 3 + 0.245x 2 + 0.2979x + 2.6872 (y: Log of MFI; x: Log of Con), R2 = 0.9999.

[0174] 2. Specificity Different cytokines were diluted to a concentration of 10 ng / mL, and their concentrations were measured according to the kit instructions. The specificity of the kit is expressed as cross-reactivity (%). The calculation method is: Cross-reactivity (%) = Detected concentration / Theoretical concentration × 100%.

[0175] The specificity detection data of the human IFN-α5 CBA kit are shown in Table 7.

[0176] Table 7. Specific detection data of human IFN-α5 CBA kit (3) Results and Discussion: Table 7 shows that the cross-reactivity rate of related cytokines is ≤0.05%. These results indicate that when using the human IFN-α5 CBA kit provided in this application to quantitatively detect human IFN-α5 in samples, it is not affected by related cytokines in the samples, demonstrating that the detection method provided in this application has good specificity.

[0177] 3. Accuracy: (1) In three different batches of experiments, each analytical batch contained three sets of test samples, and each set of test samples contained six concentrations (2, 10, 50, 250, 1250, and 6250 pg / mL). The precision of the method was expressed as the coefficient of variation (CV%), where CV% = SD / average value × 100. The accuracy was expressed as the relative error (RE%), where RE% = (average detected concentration - theoretical concentration) / theoretical concentration × 100. The accuracy of the kit was analyzed using the above parameters. The analytical results are shown in Table 8.

[0178] Table 8. Accuracy test data of human IFN-α5 CBA kit (2) Results and Discussion: Table 8 shows that the intra-assay coefficient of variation (CV) was ≤5% for all six concentration levels, and the accuracy (RE) was ≤5%. These results indicate that the quantitative detection of human IFN-α5 in samples using the human IFN-α5 CBA kit provided in this application can meet the requirements of intra-assay coefficient of variation (CV) ≤5% and accuracy (RE) ≤5%, demonstrating that the detection method provided in this application has good accuracy.

[0179] 4. Precision (1) In three different batches of experiments, each analytical batch contained three sets of test samples, and each set of test samples contained six concentrations (2, 10, 50, 250, 1250, and 6250 pg / ml). The precision of the method was expressed as the inter-batch coefficient of variation (CV%), where CV% = SD / mean value × 100%. The reproducibility of the kit was analyzed using the above parameters. The analytical results are shown in Table 9.

[0180] Table 9 Precision detection data of human IFN-α5 CBA kit (2) Results and Discussion Table 9 shows that the inter-batch coefficient of variation (CV%) for the six concentration levels (including two low, two medium, and two high concentrations) was ≤5%. These results indicate that the human IFN-α5 quantification assay using the human IFN-α5 CBA kit provided in this application can meet the requirement of an inter-batch coefficient of variation of ≤5%, demonstrating that the detection method provided in this application has good reproducibility.

[0181] 5. Sensitivity (1) In the experiments with three different kits, each analytical batch was tested 20 times for the sample dilution. The sensitivity was calculated as follows: the mean M and standard deviation SD of the measurement results were calculated, and the mean M plus twice the standard deviation SD was substituted into the standard curve in Example 4. The result obtained was the sensitivity. The analytical results are shown in Table 10.

[0182] Table 10: Experimental data on the sensitivity of the human IFN-α5 CBA detection kit. (2) Results and Discussion Table 10 shows that the sensitivity of all three batches of the kits was ≤0.5 pg / mL. These results indicate that the kit provided in this application has good sensitivity.

[0183] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An antibody against human IFN-α5 protein, characterized in that, The antibody comprises a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region and the light chain includes a light chain variable region, wherein... (a) The heavy chain variable region includes the following complementary determinant region (CDR): The amino acid sequence is CDR-H1 as shown in SEQ ID NO:2; The amino acid sequence is CDR-H2 as shown in SEQ ID NO:4; The amino acid sequence is CDR-H3 as shown in SEQ ID NO:6; and The light chain variable region includes the following complementary determinant regions (CDRs): The amino acid sequence is CDR-L1 as shown in SEQ ID NO:8; The amino acid sequence is CDR-L2 as shown in YAS; The amino acid sequence is CDR-L3 as shown in SEQ ID NO:10; or (b) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

13.

2. The antibody against human IFN-α5 protein as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:12, and The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:

14.

3. A polynucleotide encoding an antibody against human IFN-α5 protein as described in claim 1 or 2.

4. A vector containing the polynucleotide as described in claim 3.

5. A genetically engineered host cell, said host cell containing the vector as described in claim 4, or having an exogenous polynucleotide as described in claim 3 integrated into its genome.

6. An antibody conjugate comprising: (a) The antibody portion, wherein the antibody portion is an antibody against human IFN-α5 protein as described in claim 1 or 2; and (b) A conjugation portion coupled to the antibody portion, wherein the conjugation portion is a detectable marker.

7. The use of the antibody against human IFN-α5 protein as described in claim 1 or 2, or the antibody conjugate as described in claim 6, characterized in that, Reagents or kits for the preparation of human IFN-α5 protein.

8. A human IFN-α5 protein detection kit, the kit comprising an antibody against human IFN-α5 protein as described in claim 1 or 2, or an antibody conjugate as described in claim 6.

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