Anti-cd122 antibodies and methods of making and using the same

Abstract

The application relates to the field of biotechnology, in particular to an anti-CD122 antibody and preparation and application thereof. The application provides a CD122 antibody or an antigen binding fragment thereof, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1-3 as shown in SEQ ID NO: 8-10; and the CD122 antibody or the antigen binding fragment thereof further comprises a light chain variable region, wherein the light chain variable region comprises LCDR1-3 as shown in SEQ ID NO: 11-13. The application further provides biological materials, pharmaceutical compositions, kits and uses related to the CD122 antibody. The antibody provided by the application can effectively bind to a CD122 antigen and a cell membrane expressing CD122, can block the interaction between the cell membrane expressing CD122 and IL-15, and provides a new treatment scheme for IL-15, IL-2 and / or CD122 related diseases such as vitiligo and leukemia.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to an anti-CD122 antibody and its preparation and application. Background Technology

[0002] Vitiligo is a depigmenting skin disease characterized by the depletion of specific melanocytes, leading to melanin loss in the affected areas of the skin. It manifests as chronic depigmentation and milky-white lesions with well-defined borders. It is now clearly recognized as an autoimmune disease associated with metabolic and oxidative stress (including cell separation disorders), as well as genetic and environmental factors.

[0003] Cytotoxic CD8+ T cells play a central role in melanocyte elimination and contribute to the progression of vitiligo. Innate immune cells, including dendritic cells, NK cells, and ILCs, also participate, presenting melanocyte antigens or producing inflammatory cytokines (IFN-γ), respectively. Keratinocytes release inflammatory factors, including DAMPs (ATP; NLRP3) and chemokines (CXCL9 / 10 / 11 / 16), and exacerbate the disease and maintain the presence of TRMs (tissue-resident memory T cells) through IL-15-dependent survival signaling. TRM cells, characterized by CD49a, CD69, CD103, and NKG2D, persist long-term in the skin and induce vitiligo recurrence. TRMs are long-lived lymphocytes that reside in tissues and develop after the initiation of a T cell-mediated immune response. Memory T cell subsets can be recycled through circulating memory cells (TRCMs) in the blood and lymphoid organs. The high recurrence rate of vitiligo is related to the long-term presence of TRM cells in the skin. It is known that TRM cells require IL-15 to differentiate. Therefore, the IL-15 signaling pathway plays an important role in the occurrence and development of vitiligo.

[0004] IL-15 belongs to the γc cytokine family (including IL-2 / 4 / 7 / 9 / 15 and 21) and is a key factor in the regulation of lymphocyte homeostasis and function. It can promote lymphocyte proliferation and survival, thereby comprehensively enhancing the adaptive immune response. IL-15 is mainly produced by activated monocytes and dendritic cells, with a small portion produced by epithelial cells, stromal cells, and fibroblasts.

[0005] IL-15 and IL-2 share two receptor subunits, CD122 and CD132, forming a heterodimer. IL-15 signaling primarily functions through cell-to-cell communication, with CD215 trans-presenting IL-15 to the heterodimer CD122 / CD132, thereby enhancing its function. CD215 is expressed in T cells, NK cells, B cells, dendritic cells (DCs), monocytes, macrophages, and thymus and bone marrow stromal cell lines. Furthermore, CD215 mRNA has been detected in many non-lymphoid tissues, such as the liver, heart, spleen, lungs, skeletal muscle, and endothelial cells, indicating that the IL-15 / IL-15R signaling system operates in multiple tissues and organs throughout the organism.

[0006] Experiments have shown that TRMs (transmitted melanocyte receptors) exist in vitiligo lesions in humans and mice, and these TRMs also express IL-15 receptors. Melanocyte-specific T cells express CD122, while keratinocytes express CD215. It is known that TRMs require IL-15 to differentiate; therefore, blocking the IL-15 signaling pathway may inhibit the development of TRMs, thus potentially providing a long-term inhibitory effect on vitiligo. This hypothesis has been confirmed in mice, where anti-mouse CD122 antibodies have a long-term therapeutic effect on vitiligo mice, significantly reversing vitiligo symptoms, showing pigmentation, and significantly reducing TRM levels. Therefore, CD122-targeted therapy for vitiligo can provide patients with a durable treatment option, exhibiting long-term effects after a limited treatment course. Currently, there is still a need to develop superior CD122 antibodies. Summary of the Invention

[0007] To address the shortcomings of the prior art, this application presents a novel anti-CD122 antibody that can bind to the CD122 antigen and cells expressing CD122. By binding to the CD122 receptor subunit of IL-15 / IL-2, the CD122 antibody has the ability to block the interaction between CD122 or cells expressing CD122 and IL-15, thereby inhibiting the IL-15 signaling pathway and the biological processes it triggers, such as inhibiting IL-15 activation of the cell, inhibiting the proliferation of IL-15-induced leukemia cell line M07e, and inhibiting the secretion of IFNγ by IL-15-induced PBMC cells. It has therapeutic effects on diseases related to IL-15, IL-2, and / or CD122.

[0008] The first aspect of this application provides a CD122 antibody or an antigen-binding fragment thereof, including a heavy chain variable region, said heavy chain variable region including HCDR1-3 as shown in SEQ ID NO: 8-10.

[0009] The heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 14.

[0010] The CD122 antibody or its antigen-binding fragment further includes a light chain variable region, which includes LCDR1-3 as shown in SEQ ID NO: 11-13.

[0011] The light chain variable region includes an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 15.

[0012] The CD122 antibody or its antigen-binding fragment is selected from any of the following:

[0013] The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 5;

[0014] Alternatively, the heavy chain variable region may include an amino acid sequence as shown in SEQ ID NO: 14, and the light chain variable region may include an amino acid sequence as shown in SEQ ID NO: 15.

[0015] A second aspect of this application provides a polynucleotide that encodes a CD122 antibody or an antigen-binding fragment thereof as described in the first aspect above.

[0016] A third aspect of this application provides a vector comprising the polynucleotides described in the second aspect above.

[0017] A fourth aspect of this application provides a host cell containing a vector as described in the third aspect above, or the host cell's genome integrating polynucleotides as described in the second aspect above.

[0018] The fifth aspect of this application provides an antibody-drug conjugate comprising a therapeutic agent, a linker, and a CD122 antibody or an antigen-binding fragment thereof as described in the first aspect above.

[0019] The sixth aspect of this application provides a pharmaceutical composition comprising an effective amount of a CD122 antibody or an antigen-binding fragment thereof as described in the first aspect above, or an antibody-drug conjugate as described in the fifth aspect above.

[0020] The seventh aspect of this application provides a kit for detecting CD122, the kit comprising an effective amount of CD122 antibody or its antigen-binding fragment as described in the first aspect above and a detection reagent.

[0021] The eighth aspect of this application provides the use of the CD122 antibody or its antigen-binding fragment as described in the first aspect above, or the polynucleotide as described in the second aspect above, or the vector as described in the third aspect above, or the host cell as described in the fourth aspect above, or the antibody-drug conjugate as described in the fifth aspect above, or the pharmaceutical composition as described in the sixth aspect above, said use being selected from at least one of the following:

[0022] 1) To prepare products for the prevention and / or treatment of diseases related to IL-15, and / or IL-2, and / or CD122;

[0023] 2) To prepare products for the prevention and / or treatment of immune system diseases, inflammatory diseases, and tumors;

[0024] The diseases associated with IL-15, and / or IL-2, and / or CD122 are selected from vitiligo, alopecia areata, type 1 diabetes, atopic dermatitis, ulcerative colitis, systemic lupus erythematosus, eczema, psoriasis, celiac disease, graft-versus-host disease, and leukemia.

[0025] The beneficial effects of this application are as follows:

[0026] The CD122 antibody of this application can effectively bind to the CD122 antigen and cells expressing CD122 on their cell membranes, and can block the interaction between cells expressing CD122 and IL-15. This indicates that the CD122 antibody of this application can inhibit the binding of IL-15 or IL-2 to its receptor CD122, thereby inhibiting IL-15 and / or IL-2 related signaling pathways. It is expected that it can treat diseases related to IL-15, and / or IL-2, and / or CD122 signaling pathways. Furthermore, this application verifies that the developed CD122 antibody has an inhibitory effect on IL-15-activated cell membrane-expressing CD122 cells, inhibits the proliferation of CD122-expressing tumor cells, and inhibits the secretion of anti-inflammatory factors by peripheral blood mononuclear cells expressing CD122. The effect is superior to the MAB05 antibody in the prior art. This application provides a new treatment option for diseases related to IL-15, and / or IL-2, and / or CD122, such as vitiligo, alopecia areata, type 1 diabetes, atopic dermatitis, ulcerative colitis, systemic lupus erythematosus, eczema, psoriasis, celiac disease, graft-versus-host disease, leukemia, etc. Attached Figure Description

[0027] Figure 1 The results show the binding activity of the murine antibody to the hCD122-His protein in Example 2.

[0028] Figure 2 This is the result of the cell-binding activity of the mouse antibody in Example 2.

[0029] Figure 3 This is the result of the cell blocking activity of the mouse antibody in Example 2.

[0030] Figure 4 This is the result of the chimeric antibody cell blocking activity in Example 3.

[0031] Figure 5 This is the result of the cell-binding activity of the humanized antibody in Example 4.

[0032] Figure 6 This is the result of the cell blocking activity of the humanized antibody in Example 4.

[0033] Figure 7 This is the result of the humanized antibody inhibiting IL-15-activated cells in Example 4.

[0034] Figure 8 This is the result of the humanized antibody inhibiting IL-15-induced M07e cell proliferation in Example 4.

[0035] Figure 9 This is the result of the humanized antibody inhibiting the secretion of IFNγ by PBMC cells in Example 4. Detailed Implementation

[0036] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application.

[0037] Before further describing the specific embodiments of this application, it should be understood that the scope of protection of this application is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of this application is for describing specific embodiments and not for limiting the scope of protection of this application; in the specification and claims of this application, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0038] This application first provides a CD122 antibody or an antigen-binding fragment thereof.

[0039] Antibodies typically exist as one or more Y-shaped monomers, each consisting of four polypeptide chains: two identical heavy chains and two identical light chains. The heavy and light chains are distinguished by their molecular weight. Based on differences in the structure of small polypeptide molecules, light chains are classified as κ or λ types, and heavy chains as μ, δ, γ, α, or ε, defining the antibody class or type as IgM, IgD, IgG, IgA, and IgE, respectively.

[0040] The variable region of an antibody is the region in the antibody molecule responsible for binding to a specific antigenic epitope. It determines the antigen specificity of the antibody. The variable region includes the heavy chain variable region and the light chain variable region. The heavy chain variable region (VH) is located at the N-terminus of the antibody heavy chain (H chain), and the light chain variable region (VL) is located at the N-terminus of the antibody light chain (L chain). These regions together constitute the antigen-binding site of the antibody.

[0041] The variable region includes complementarity-determining regions (CDRs), which are key regions in the antibody molecule that specifically bind to antigens. CDR regions exhibit high sequence diversity, enabling antibodies to recognize and bind to a wide variety of antigens. In the antibody's variable region, both the light and heavy chains have three CDR regions, denoted as CDR1, CDR2, and CDR3, respectively. CDR regions directly interact with specific epitopes of the antigen, and their sequence diversity determines the antibody's specificity and affinity. In the antibody's heavy chain variable region, CDR regions are typically located at specific amino acid positions.

[0042] The CD122 antibody or its antigen-binding fragment includes a heavy chain variable region, which includes HCDR1 as shown in SEQ ID NO: 8, HCDR2 as shown in SEQ ID NO: 9, and HCDR3 as shown in SEQ ID NO: 10.

[0043] The heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 14.

[0044] The CD122 antibody or its antigen-binding fragment further includes a light chain variable region, which includes LCDR1 as shown in SEQ ID NO: 11, LCDR2 as shown in SEQ ID NO: 12, and LCDR3 as shown in SEQ ID NO: 13.

[0045] The light chain variable region includes an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 15.

[0046] HCDR1 (SEQ ID NO: 8): DYEMH.

[0047] HCDR2 (SEQ ID NO: 9): AIDPETGGTDYNQKFKG.

[0048] HCDR3 (SEQ ID NO: 10): SYDYGVDWYFDV.

[0049] LCDR1 (SEQ ID NO: 11): RSSQSLVHSNGNTYLH.

[0050] LCDR2 (SEQ ID NO: 12): KVSNRFS.

[0051] LCDR3 (SEQ ID NO: 13): SQSTHVPYT.

[0052] In a specific embodiment of this application, the heavy chain variable region of the CD122 antibody includes the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 5, for example, the murine CD122 antibody 589E2D4 in this application.

[0053] Heavy chain variable region of 589E2D4 (SEQ ID NO: 3): QVQLQQSGAELVRPGASVTLSCKASGYT FTDYEMHWVKQTPVHGLEWIGAIDPETGGTDYNQKFKGKATLTADKSSSTAYMELRSLTSE DSAVYYCTMSYDYGVDWYFDVWGAGTTVTVSS

[0054] Light chain variable region of 589E2D4 (SEQ ID NO: 5): DVVMTQIPLSLPVSLGDQASISCRSSQSLV HSNGNTYLHWYLQKPGQSPNLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIK

[0055] In another specific embodiment of this application, the heavy chain variable region of the CD122 antibody includes the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 15. For example, in this application, the heavy chain CDR region of the murine CD122 antibody 589E2D4 is replaced with the heavy chain CDR region of IGHV1-3*01, and the light chain CDR region of the murine CD122 antibody 589E2D4 is replaced with the light chain CDR region of IGKV2-28*01 to obtain the humanized CD122 antibody 589-81.

[0056] Heavy chain variable region of 589-81 (SEQ ID NO: 14): QVQLVQSGAEVKKPGASVKVSCKASGYTF TDYEMHWVRQAPGQRLEWMGAIDPETGGTDYNQKFKGRVTITRDTSASTAYMELSSLRSE DTAVYYCTMSYDYGVDWYFDVWGQGTTVTVSS.

[0057] Light chain variable region of 589-81 (SEQ ID NO: 15): DIVMTQSPLSLPVTPGEPASISCRSSQSLVH SNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIK.

[0058] In a specific embodiment of this application, the heavy chain variable region and / or light chain variable region further include amino acid mutations; preferably, the amino acid mutations are located in the FR framework region.

[0059] The FR (Framework Region) refers to the region within the antibody variable region excluding the three CDRs. Its main function is to stabilize the spatial conformation of the CDR region and assist in the accurate and specific binding of the antigen. Each antibody chain has four framework regions: FR1, FR2, FR3, and FR4. The relatively conserved amino acid sequences of the framework regions form a β-sheet structure, supporting the CDR region located at the top of the barrel structure, enabling the CDR region to bind to the antigen epitope.

[0060] In this application, the heavy chain variable region is selected from amino acid sequences that have at least 99%, 95%, 90%, or 85% identity with and have the same function as SEQ ID NO: 3, 14; and / or, the light chain variable region is selected from amino acid sequences that have at least 99%, 95%, 90%, or 85% identity with and have the same function as SEQ ID NO: 5, 15.

[0061] In addition to variable regions, the heavy and light chains of the antibody may also include constant regions. The constant regions may be human or mouse-derived. The constant regions of the antibody heavy chain may be IgG, IgA, IgM, IgD, or IgE heavy chain constant regions, and the constant regions of the antibody light chain may be Kappa light chain constant regions or Lambda light chain constant regions.

[0062] In this application, the heavy chain constant region and the light chain constant region may also include amino acid mutations, such as different subtypes of IgG and IgA.

[0063] For example, in some embodiments of this application, the heavy chain constant region of the antibody is the heavy chain constant region of human antibody IgG1, and the light chain constant region is the light chain constant region of human Kappa.

[0064] In a specific embodiment of this application, the heavy chain variable region sequence of 589-81 is linked to the human IgG1 subtype heavy chain constant region to obtain the heavy chain sequence of 589-81 (SEQ ID NO: 16):

[0065] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQRLEWMGAIDPETGGTDYNQKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCTMSYDYGVDWYFDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0066] In a specific embodiment of this application, the variable region sequence of the light chain 589-81 is connected to the constant region of the light chain of the kappa subtype to obtain the light chain sequence 589-81 (SEQ ID NO: 17):

[0067] DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0068] In specific embodiments of this application, the antibody is selected from polyclonal antibodies, monoclonal antibodies, single-chain antibodies, antigen-binding domains, bispecific antibodies, multispecific antibodies, or antigen-binding portions of chimeric antigen receptors.

[0069] Those skilled in the art will recognize that antibodies in non-complete tetrameric forms, including but not limited to Fab, Fab', F(ab') or F(ab')2, nanobodies (VHH), single-chain antibodies (scFv), BsFv, dsFv, (dsFv)2, or Fv, can also exert the effect of specifically binding antigens. Therefore, this application also includes antigen-binding fragments of antibodies.

[0070] In specific embodiments of this application, the antibody is selected from murine antibodies, chimeric antibodies, and humanized antibodies.

[0071] Specifically, murine antibodies are antibodies whose encoding genes are entirely derived from mice. Chimeric antibodies are monoclonal antibodies produced by inserting the variable regions of the light and heavy chains of murine monoclonal antibodies into a vector containing the constant regions of human antibodies using DNA recombination technology, followed by expression in mammalian cells. Chimeric antibodies can achieve a humanization rate of up to 70%, fully retaining the variable regions and parental activity of the murine monoclonal antibody, while the introduction of the human antibody constant regions reduces immunogenicity. Humanized antibodies are antibodies modified through genetic engineering, with their structure largely or entirely composed of human antibody sequences to reduce immunogenicity in humans. This modification typically involves transplanting the complementarity-determining regions (CDRs) of murine monoclonal antibodies into the framework regions (FRs) of human antibodies, while retaining the antigen-binding sites of the murine antibody. This results in modified antibodies with lower immunogenicity in humans while maintaining their original specificity and affinity.

[0072] This application also provides a polynucleotide encoding the above-described CD122 antibody or its antigen-binding fragment.

[0073] Polynucleotides are polymers of nucleotides typically linked from one deoxyribose or ribose to another. The polynucleotides used in this application are not limited in size and may include polynucleotides containing modifications, particularly modified nucleotides. In some embodiments, the polynucleotide may be RNA, DNA, or cDNA, etc. The preparation methods of the polynucleotides are existing technologies, and conventional methods can be selected according to specific circumstances. For example, they can be prepared by automated DNA synthesis, recombinant DNA technology, or isolated from suitable natural sources.

[0074] In a specific embodiment of this application, the polynucleotide includes nucleotide sequences as shown in SEQ ID NO: 2 and 4, which are nucleotide sequences encoding the murine antibody 589E2D4.

[0075] Nucleotide sequence of the heavy chain variable region of 589E2D4 (SEQ ID NO: 2): CAGGTTCAACTGCAGCAGT CTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCTATTGATCCTGAAACTGGTGGTACTGACTACAATCAGAAATT CAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAATGTCCTATGATTACGGGGTGGACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

[0076] Nucleotide sequence of the light chain variable region of 589E2D4 (SEQ ID NO: 4): GATGTTGTGATGACCCAAA TTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTTCAGAAGCCAGGCCAGTCTCCAAATCTCCTGATCTATAAAGTTTCCAACCGA TTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0077] Based on the above-mentioned polynucleotides, the polynucleotides can be inserted into a vector using existing technology to enable their expression. Therefore, this application also provides a vector containing the above-mentioned polynucleotides.

[0078] In this application, "vector" refers to a polynucleotide capable of carrying at least one polynucleotide fragment. The vector can exist in a circular or linear (linearized) form and includes vector fragments, or it can be an artificial chromosome or similar polynucleotide containing a transferable exogenous nucleic acid fragment. The vector may contain at least one expression cassette containing a regulatory sequence for the proper expression of the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (e.g., a polynucleotide encoding a target product or a selectable marker) can be inserted into the expression cassette of the vector for expression therefrom. The vector can also integrate exogenous DNA sequences into the host cell's genome, thereby achieving stable expression of the exogenous gene. It typically contains homologous arms or specific DNA sequences that can undergo homologous recombination with specific locations in the host cell's genome. The vector may be selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors, etc.

[0079] Based on the above-described polynucleotides and vectors, this application may also provide a host cell containing the above-described vectors or having the above-described polynucleotides integrated into its genome.

[0080] The host cells can be selected from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc. Specifically, they can be selected from Escherichia coli, Streptomyces, Salmonella typhimurium, yeast, filamentous fungi, Drosophila S2 or Sf9 cells, CHO cells, COS cells, HEK293F cells, Bowes melanoma cells, NS0 cells, BHK cells, PER.C6 cells, etc.

[0081] Furthermore, the host cell is a cell in which a vector containing the aforementioned polynucleotide has been introduced. The method for introducing the vector into the host cell can be conventional, depending on the actual situation. For example, methods such as microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, liposome transfection, and PEI can be selected.

[0082] According to the method for preparing antibodies by genetic engineering, this application also provides a method for preparing CD122 antibody or its antigen-binding fragment, comprising: culturing a host cell containing the above-mentioned vector or a host cell whose genome has integrated exogenous polynucleotides, under conditions that allow the expression of the CD122 antibody or its antigen-binding fragment, and recovering the CD122 antibody or its antigen-binding fragment from the cultured host cell culture.

[0083] The conditions that allow the expression of the CD122 antibody or its antigen-binding fragment typically refer to effective transfection or transduction methods, favorable promoter conditions, stable gene copies, suitable culture conditions, and optimized post-translational modifications.

[0084] The effective transfection or transduction methods can be selected from microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, liposome transfection, PEI, and other methods.

[0085] The recovery of the CD122 antibody or its antigen-binding fragment can be carried out using conventional methods, such as Protein A affinity chromatography purification, depending on the actual situation.

[0086] This application may also provide an antibody-drug conjugate comprising a therapeutic agent, a linker, and the aforementioned CD122 antibody or its antigen-binding fragment.

[0087] Antibody-drug conjugates (ADCs) are small molecule drugs that are linked to monoclonal antibodies via a chemical chain. The monoclonal antibody then acts as a carrier to target and deliver the drug to target cells. Their general structural formula can be A-(LU)n, where: A is an antibody or its antigen-binding fragment; U is a therapeutic agent; L is a linker; and n is an integer selected from 1 to 8.

[0088] Antibody-drug conjugates include linkers that conjugate the antibody or its antigen-binding fragment to the therapeutic agent. The linkers can be cleavable linkers or non-cleavable linkers. Cleavable linkers can include acid-cleavable linkers, reducible linkers, enzyme-cleavable linkers, etc., while non-cleavable linkers are divided into two categories: thioether or maleimide hexanoyl (MC).

[0089] The therapeutic agents refer to small molecule drugs with cytotoxicity that are covalently coupled to antibodies via linkers. These therapeutic agents are the core components of ADCs and are responsible for exerting anti-tumor effects. Examples include microtubule inhibitors, DNA damaging agents, topoisomerase inhibitors, apoptosis inducers, immunomodulators, and protein degraders. Microtubule inhibitors can be, for example, maytansine derivatives (DM1, DM3, DM4), monomethylolpropionate E (MMAE), paclitaxel, and eribulin. DNA damaging agents can be, for example, calicheamicin, duocarmysin, atrazocin-like drugs (PBD), camptothecins, and camptothecin derivatives (SN-3). Topoisomerase inhibitors can be, for example, DXd (Deruxtecan), SN-38, and Exatecan (DX-895). Immunomodulators can be, for example, TLR agonists, STING agonists, and BTK inhibitors.

[0090] This application also provides a pharmaceutical composition comprising an effective amount of the above-mentioned CD122 antibody or its antigen-binding fragment, or the above-mentioned antibody-drug conjugate, for the prevention and / or treatment of IL-15, and / or IL-2, and / or CD122-related diseases.

[0091] The effective amount refers to the amount of a pharmaceutical compound or composition that causes a measurable clinical, biological, or pharmaceutical change or response in a biomarker, cell, tissue, system, or patient.

[0092] The form of the pharmaceutical composition is not limited and can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0093] In specific embodiments of this application, the pharmaceutical composition may further include pharmaceutically acceptable excipients or carriers. Pharmaceutically acceptable refers to non-toxic materials that do not interfere with the bioactivity and effectiveness of the active ingredient. Specific examples of pharmaceutically acceptable excipients or carriers include sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders, etc. Furthermore, it may also contain other low-molecular-weight peptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine, and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; and sugar alcohols such as mannitol or sorbitol. When preparing aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other excipients like D-sorbitol, D-mannose, D-mannitol, or sodium chloride, appropriate solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), and nonionic surfactants (Tween 80, HCO-50) can be used. The excipients or carriers can be adjusted according to the desired dosage form, and the ratio can be adjusted based on actual needs.

[0094] In specific embodiments of this application, the antibody or antibody-drug conjugate in the pharmaceutical composition can be a single active ingredient, or it can be combined with one or more other active ingredients that have therapeutic effects on IL-15, and / or, and / or CD122-related diseases to form a combined preparation. The efficacy and safety of each component do not conflict. For example, other active ingredients for treating vitiligo include Compound Deworming Chrysanthemum Pills, Vitiligo Pills, Relapse Babuqi Tablets, Wulong Powder, Astragalus and Jujube Oral Liquid, Peach Blossom Serum Pills, Relapse Muniziqi Granules, Deworming Chrysanthemum Injection, Psoralea Injection, Calf Spleen Extract Injection, Calf Serum Protein-Free Injection, photosensitizers, corticosteroids, topical antioxidants, glucocorticoid preparations (such as halometasone, mometasone furoate), calcineurin inhibitors (such as tacrolimus ointment or bimetalimus ointment), vitamin D3 derivatives (such as calcipotriol or tacalcitrol), nitrogen mustard ethanol, nitrogen mustard hydrochloride, promethazine, and JAK inhibitors (ruxolitinib). For example, other active ingredients for treating leukemia include cytarabine, cyclophosphamide, methotrexate, daunorubicin, imatinib mesylate tablets, and dasatinib. The content of each component in the combination preparation is usually a safe and effective amount, which can be adjusted based on actual usage (e.g., patient weight, type of application, disease condition, severity).

[0095] This application also provides a kit for detecting CD122, the kit comprising an effective amount of the above-mentioned CD122 antibody or its antigen-binding fragment and a detection reagent, for detecting the content of CD122 in a sample.

[0096] The detection reagent may be selected from any one or more of enzyme-linked immunosorbent assay (ELISA) reagents, cell immunofluorescence assay reagents, flow cytometry reagents, isotope tracers, contrast agents, magnetic nanoparticles, or imaging agents.

[0097] IL-15 and IL-2 are important cytokines with very similar structures, both belonging to the helical cytokine family, and closely related to CD122, which is a common receptor subunit for both IL-15 and IL-2. Due to these shared receptor components and the shared JAK1 / JAK3 / STAT5 signaling pathway, IL-15 and IL-2 have similar functions, including stimulating T cell proliferation, producing cytotoxic T lymphocytes, stimulating B cell immunoglobulin synthesis, and the production and sustained survival of NK cells. Therefore, based on the structural and functional similarities of IL-15 and IL-2, and considering the effects of the CD122 antibody of this application on the IL-15 signaling pathway and IL-15-related diseases, it can be concluded that it has similar effects on the IL-2 signaling pathway or the IL-15 / IL-2 signaling pathway and related diseases.

[0098] This application also provides the use of the above-described CD122 antibody or its antigen-binding fragment, or the above-described polynucleotide, or the above-described carrier, or the above-described host cell, or the above-described antibody-drug conjugate, or the above-described pharmaceutical composition, wherein the use is selected from at least one of the following:

[0099] 1) To prepare products for the prevention and / or treatment of diseases associated with IL-15, and / or IL-2, and / or CD122;

[0100] 2) Prepare products for the prevention and / or treatment of immune system diseases, inflammatory diseases, and tumors.

[0101] The IL-15 receptor consists of three subunits: the α chain (IL-15Rα), the β chain (CD122, also known as IL-2 / IL-15Rβ), and the γ chain (CD132, also known as γc). CD122 is a key subunit of the IL-15 receptor, shared with the IL-2 receptor. After binding to the IL-15Rα subunit, IL-15 remains on the cell surface and forms an immune synapse with IL-2R / IL-15Rβ-γc on nearby effector NK cells, T cells, or tumor cells. This activates the JAK1 / JAK3 and STAT3 / STAT5 pathways, Syk kinase and phospholipase C (PLC)γ, Lck kinase, and Shc, leading to the activation of the PI3K / Akt and Ras / Raf / MAPK signaling cascades. The CD122 antibody of this application binds to the receptor subunit CD122 of IL-15, thereby blocking the binding of IL-15 to its receptor subunit CD122, thus blocking the IL-15 signaling pathway and its downstream signaling pathways and the resulting biological processes, and has a therapeutic effect on diseases related to IL-15, and / or IL-2, and / or CD122.

[0102] The diseases associated with IL-15, and / or IL-2, and / or CD122 are selected from vitiligo, alopecia areata, type 1 diabetes, atopic dermatitis, ulcerative colitis, systemic lupus erythematosus, eczema, psoriasis, celiac disease, graft-versus-host disease, and leukemia.

[0103] The leukemias mentioned include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), among which acute myeloid leukemia includes granulocytic leukemia, monocytic leukemia, and megakaryoblastic leukemia.

[0104] In this application, the tumor includes solid tumors and non-solid tumors, and the tumor cells of the tumor express CD122.

[0105] The product has one or more of the following effects:

[0106] 1) Inhibits IL-15 / IL-2 activation of cells;

[0107] 2) Inhibits IL-15 / IL-2-mediated tumor cell proliferation;

[0108] 3) Inhibits the secretion of pro-inflammatory factors by immune cells mediated by IL-15 / IL-2;

[0109] The cells, tumor cells, and immune cells mentioned above express CD122.

[0110] The product may include an effective dose of the above-mentioned ingredients as the main active ingredient, or may be prepared from the above-mentioned ingredients.

[0111] The products include, but are not limited to, drugs, drug compositions, combinations of drugs, reagents, and kits.

[0112] The dosage form of the product is not limited, and may be one or more of the following: solution, injection, spray, nasal drops, aerosol, powder spray, tablet, capsule and granule.

[0113] The product may also include pharmaceutically acceptable carriers or excipients, as described above. The product may be administered via the gastrointestinal tract or non-gastrointestinal route, such as by injection, spray, nasal drops, eye drops, osmosis, absorption, or physical or chemically mediated methods into the body, including intramuscular, intradermal, subcutaneous, intravenous, and mucosal tissues; or by being mixed with or encapsulated by other substances before being administered into the body. Preferably, it is administered via the gastrointestinal tract. Therefore, preferably, the pharmaceutical composition further includes antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the subject's blood, as well as a sterile suspension containing both aqueous and non-aqueous components, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0114] This application also provides a method for treating diseases related to IL-15, and / or IL-2, and / or CD122, comprising administering to a subject an effective amount of the aforementioned CD122 antibody or its antigen-binding fragment, or the aforementioned antibody-drug conjugate, or the aforementioned pharmaceutical composition, or the aforementioned product.

[0115] The diseases associated with IL-15, and / or IL-2, and / or CD122 are selected from vitiligo, alopecia areata, type 1 diabetes, atopic dermatitis, ulcerative colitis, systemic lupus erythematosus, eczema, psoriasis, celiac disease, graft-versus-host disease, and leukemia.

[0116] In this application, the target of application of the aforementioned CD122 antibody or its antigen-binding fragment, antibody-drug conjugate, pharmaceutical composition, kit, product, or method can be a mammal, such as, but not limited to, humans, primates, livestock (e.g., sheep, cattle, horses, donkeys, pigs), pets (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), or captured wild animals (e.g., foxes, deer); preferably, the target is a primate; more preferably, the target is a human.

[0117] In this application, the aforementioned CD122 antibody or its antigen-binding fragment, antibody-drug conjugate, pharmaceutical composition, product, and method may be used alone or in combination with other methods for treating IL-15, and / or IL-2, and / or CD122-related diseases, or other methods for blocking IL-15, and / or IL-2, and / or CD122 signaling pathways, such as targeted drug therapy, chemotherapy drug therapy, and surgical treatment.

[0118] The following will illustrate the specific implementation through examples. When numerical ranges are given in the examples, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, based on the knowledge of the prior art possessed by those skilled in the art and the description in this application, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the examples of this application may be used to implement this application. Unless otherwise specified, the instruments, materials, and reagents used in the examples are obtained through conventional means.

[0119] Experimental materials:

[0120] Mouse myeloma cells SP2 / 0: purchased from ATCC, catalog number CRL-1581.

[0121] Balb / c mice: purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0122] HRP-Goat Anti-Mouse Secondary Antibody: Purchased from Millipore, catalog number AP181P.

[0123] HRP-goat anti-human IgG Fc secondary antibody: purchased from Sigma, product number A0170-1ML.

[0124] Donkey anti-mouse PE fluorescent secondary antibody: purchased from Jackson, catalog number 715-116-150.

[0125] Goat anti-human PE fluorescent secondary antibody: purchased from Jackson, catalog number 109-115-098.

[0126] TMB: Purchased from KPL Company, item number 52-00-03.

[0127] Bovine serum albumin (BSA): purchased from Sangon Biotech, product number A600332-0100.

[0128] RPMI 1640 Medium: Purchased from Gibco, part number 61870127.

[0129] DMEM: Purchased from Gibco, part number 11995065.

[0130] GM-CSF: Purchased from E-Tech, item number 10015-HNAH.

[0131] Penicillin-streptomycin: Purchased from Gibco, catalog number 15140122.

[0132] Fetal bovine serum (FBS): purchased from Gibco, catalog number 10091-148.

[0133] Polyethylene glycol solution was purchased from Sigma, product number P7181.

[0134] Hybridoma-SFM was purchased from life technologies, item number 12045-076.

[0135] HAT: Purchased from Gibco, item number 21060017.

[0136] pcDNA3.4: Purchased from ThermoFisher, catalog number A14697.

[0137] EZ-Link NHS-Biotin Reagent: Purchased from Thermo Fisher, part number 20217.

[0138] HBS-EP pH7.4 buffer solution: purchased from GE Healthcare, catalog number BR-1006-69.

[0139] Protein A / G chip: purchased from GE Healthcare, catalog number BR-1005-30.

[0140] Bio-Glo fluorescent reagent: purchased from Promega, catalog number G7940.

[0141] CCK8: Purchased from Dojindo, item number CK04.

[0142] IL-15: Purchased from E-Cho Co., Ltd., item number 10360-H07E.

[0143] Human IL-2R beta / IL-2R gamma(Luc)HEK293 Reporter Cell: Purchased from ACRO, catalog number CHEK-ATF136.

[0144] Puromysin: Purchased from Invivogen, product number ant-pr-5b.

[0145] Zeocin: Purchased from Invivogen, product number R25001.

[0146] G418: Purchased from Invivogen, product number ant-gn-5.

[0147] Human IFNγELISAPair Set: Purchased from E.K. Inc., item number SEKA11725.

[0148] Example 1: Preparation and screening of cell-immunized animals and hybridomas

[0149] 1.1 Antigen Expression

[0150] The extracellular region gene of human CD122 (sequence from UniProt, accession number P14784, as shown in SEQ ID NO.1) was constructed into the pcDNA3.4 expression vector using conventional gene synthesis and molecular cloning methods. A signal peptide sequence was added to its N-terminus and a 6×His tag was added to its C-terminus. HEK-293F cells were transfected and expressed for 5 days. The cell culture supernatant was collected and purified to obtain hCD122-His protein.

[0151] Human CD122 extracellular amino acid sequence (hCD122-ECD(27-240)):

[0152] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAP ISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT(SEQ IDNO: 1).

[0153] 1.2 Cell-immunized mice

[0154] Balb / c mice were immunized with cells that highly expressed CD122 on their cell membranes (Human IL-2R beta / IL-2R gamma(Luc)HEK293Reporter Cell, purchased from ACRO, catalog number CHEK-ATF136): The cells used for immunization were first washed twice with PBS, then resuspended to 1.5e7 cells / mL, and then 300 mL (i.e., 5e6 cells per mouse) was injected intraperitoneally into each mouse. Immunization was repeated every two weeks. After four immunizations, 50 μg / mouse / 0.2 mL of soluble hCD122-His protein was injected intraperitoneally to stimulate immunization. 3-4 days later, the spleens of the mice were harvested for fusion experiments.

[0155] 1.3 Preparation and Screening of Hybridomas

[0156] Three to four days after the final immunization of mice, mouse spleen cells were PEG-fused with mouse myeloma cells SP2 / 0 using a standard hybridoma technique. The fused cells were then uniformly suspended in complete culture medium consisting of RPMI 1640-GLUMAX with 1% penicillin-streptomycin, 20% FBS, and 1% HAT. The fused cells were seeded at a rate of 3*10⁴ cells / 200 μl / well in 60 96-well cell culture plates and cultured in an incubator. After 7-12 days, the supernatant was harvested, and hybridoma wells showing positive human CD122 binding activity were screened using ELISA.

[0157] The method for screening hybridoma wells with positive human CD122 binding activity using ELISA is as follows: hCD122-His is diluted to 1 μg / ml with PBS buffer, and 100 μl / well is added to the ELISA plate. The plate is incubated overnight at 4°C. The supernatant is discarded the next day, the plate is washed once with PBST, and 5% skim milk powder (prepared with PBS) is added. The plate is blocked at 37°C for 1 hour, and then washed three times with PBST. The collected hybridoma supernatant is then added sequentially to the blocked ELISA plate, 100 μL / well, and incubated at 37°C for 1 hour. Wash the plate three times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody, and incubate at 37°C for 1 hour. After washing the plate three times with PBST, blot dry any remaining droplets on absorbent paper, add 100 μL of TMB to each well, and incubate at room temperature in the dark for 5 minutes. Stop the substrate reaction by adding 50 μL of 2M H2SO4 to each well, and read the OD value at 450 nm using a multi-functional microplate reader to analyze the binding ability of the antibody to the target antigen hCD122-His. Candidate hybridoma cell lines were then selected. The hybridoma cell lines were amplified and screened in serum-containing complete medium, centrifuged, and transferred to serum-free Hybridoma-SFM medium to maintain a cell density of 1–2 × 10⁶ cells / well. 7 / ml, cultured at 8% CO2 and 37℃ for 1 week, centrifuged to obtain the culture supernatant, purified by Protein G affinity chromatography to obtain various mouse monoclonal antibody proteins against human CD122 and named them respectively.

[0158] Example 2: Detection of mouse antibody activity

[0159] 2.1 Binding activity of murine antibody against hCD122-His protein

[0160] The binding activity of mouse antibodies to hCD122-His protein was determined by indirect enzyme-linked immunosorbent assay (ELISA). The specific method is as follows:

[0161] Dilute hCD122-His protein to 1 μg / mL in coating buffer (50 mM carbonate coating buffer, pH 9.6) and coat ELISA plates overnight at 4°C. Discard the supernatant, wash the plates three times with PBST, and then block with 5% skim milk powder prepared in PBS, incubating at 37°C for 1 h. After washing once with PBST, serially dilute the prepared anti-human CD122 mouse monoclonal antibodies with 1% BSA buffer prepared in PBST, adding 100 μL / well to the above ELISA plates, and incubating at 37°C for 1 h. Wash the plates three times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody, and incubate at 37°C for 1 h. After washing three times with PBST, blot dry any remaining droplets on absorbent paper, add 100 μL of TMB chromogenic solution to each well, and develop at room temperature in the dark for 5 min. Add 50 μL of 2M chromogenic solution to each well. The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450 nm using a multi-functional microplate reader to analyze the binding ability of the test antibody to the target antigen hCD122-His. The experimental results are as follows: Figure 1 As shown.

[0162] 2.2 Cell-binding activity of murine antibodies

[0163] Cell binding activity assay: Human IL-2R beta / IL-2R gamma(Luc)HEK293 ReporterCell cells were digested, counted, resuspended, and seeded at 2e5 cells / well in a U-bottom 96-well plate. The cells were washed twice with 1% BSA buffer prepared with PBST, and the supernatant was discarded. Mouse monoclonal antibody was serially diluted with 1% BSA buffer prepared with PBST, and 100 μL / well was added to the U-bottom 96-well plate. The mixture was thoroughly mixed and incubated at 4°C for 1 h. After centrifugation, the supernatant was discarded, and the cells were washed twice with 1% BSA buffer prepared with PBST. 100 μL / well was then added to the plate with a 1:500 dilution of anti-mouse IgG-PE (anti-human IgG). IgG-PE secondary antibody was mixed thoroughly and incubated at 4°C for 1 hour. After centrifugation, the supernatant was discarded, and the cells were washed twice with 1% BSA buffer prepared with PBST. The final volume was adjusted to 200 μL / well. Fluorescence intensity was detected by flow cytometry to analyze the affinity of the antibody for cells. Representative experimental results are shown below. Figure 2 As shown.

[0164] 2.3 Cell blocking activity of murine antibody

[0165] Cell blockade activity assay: Human IL-2R beta / IL-2R gamma(Luc)HEK293 ReporterCell cells were digested, counted, resuspended, and seeded at 2e5 cells / well in U-bottom 96-well plates. The cells were washed twice with 1% BSA buffer prepared with PBST, and the supernatant was discarded. Mouse monoclonal antibodies were serially diluted with 1% BSA buffer prepared with PBST, and 100 μL / well was added to the U-bottom 96-well plates. The mixture was thoroughly mixed and incubated at 4°C for 1 h. After centrifugation, the supernatant was discarded, and the cells were washed twice with 1% BSA buffer prepared with PBST. After centrifugation, the supernatant was discarded, and hIL-15-647 was diluted to 100 ng / mL with 1% BSA buffer prepared with PBST. The mixture was added to the U-bottom 96-well plates at 100 μL / well, thoroughly mixed, and incubated at 4°C for 1 h. After centrifugation, the supernatant was discarded, and the cells were washed twice with 1% BSA buffer prepared with PBST. The final volume was adjusted to 200 μL / well. Fluorescence intensity was detected by flow cytometry to analyze the blocking ability of the test antibody against the interaction between cells and hIL-15. The obtained data were analyzed using GraphPad Prism 9 software. Experimental results are as follows: Figure 3 As shown.

[0166] In summary Figures 1-3 The results showed that the two murine antibodies, 401C10D4 and 589E2D4, had the best activity.

[0167] Example 3: Humanization of mouse-derived anti-human CD122 monoclonal antibody

[0168] 3.1 Determination of the variable region sequence of murine anti-human CD122 monoclonal antibody

[0169] Hybridoma clones 401C10D4 and 589E2D4 were selected as candidate antibodies. Total RNA was extracted from the corresponding hybridoma monoclonal cell lines using Trizol (purchased from Lifetechnologies). The mRNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Takara). PCR was performed using a primer combination reported in the literature (Antibody Engineering, Volume 1, edited by Roland Kontermann and Stefan Dübel, primer sequence from page 323). The obtained PCR product was sequenced and analyzed using the Kabat database to confirm that the obtained sequence was the variable region sequence of the murine antibody.

[0170] The full-length heavy chain variable region gene sequence of 589E2D4 is 363 bp, encoding 121 amino acid residues. The nucleotide sequence is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 3. The full-length light chain variable region gene sequence is 336 bp, encoding 112 amino acid residues. The nucleotide sequence is shown in SEQ ID NO: 4, and the amino acid sequence is shown in SEQ ID NO: 5.

[0171] The aforementioned antibodies can be isolated monoclonal antibodies through conventional genetic engineering. For example, the gene for synthesizing the aforementioned antibodies can be used to construct a recombinant vector, which can then be transfected into 293 cells for recombinant expression, followed by purification.

[0172] 3.2 Construction, expression, and validation of chimeric antibody blocking activity

[0173] The murine antibodies 401C10D4 and 589E2D4 obtained in Example 3.1, as well as the heavy and light chain variable regions of the positive control antibody MAB05, were linked to the human IgG1 subtype constant region and the kappa subtype light chain constant region, respectively, and cloned into the pcdna3.4 vector. The recombinant plasmid was then extracted and co-transfected into CHO cells and / or 293F cells. After 5-7 days of cell culture, the culture medium was centrifuged at high speed, filtered under vacuum through a microporous membrane, and then loaded onto a HiTrap MabSelectSuRe column. Proteins were eluted in one step with elution buffer containing 100 mM citric acid and pH 3.5. The target sample was recovered and dialyzed against PBS at pH 7.4 to obtain chimeric antibodies C-401C10 and C-589E2, as well as the positive control antibody MAB05. Following the "Cell Blocking Activity Assay" in Example 2, the blocking activity of the chimeric antibodies and the positive control antibody MAB05 on the interaction between Human IL-2R beta / IL-2R gamma (Luc) HEK293 Reporter Cell cells and human IL-15 was compared.

[0174] Experimental results are as follows Figure 4 As shown, the chimeric antibody C-589E2 exhibits superior cell blocking activity compared to the positive control antibody MAB05. The sequence of the positive control antibody MAB05 is derived from patent CN117295762A.

[0175] MAB05-VH (SEQ ID NO: 6):

[0176] EVQLLESGGGLVQPGGSLRLSCAASGFTVTSYAVHWVRQAPGKGLEWLGVIWSGGSTDYNAAFISRLTISKDNSKNTVYFQMNSLRAEDTAVYYCARAGDANYDGFAYWGQGTLVTVSS

[0177] MAB05-VL (SEQ ID NO: 7):

[0178] DIQMTQSPSSSLSASVGDRVTITCQASQSVSFLYWYQQRPGKAPRRLLIYDTSNLASGVPS RFSGSGSGTSYTFTISSLQPEDIATYYCQQWSTYPLTFGQGTKVEIK

[0179] Example 4: Detection of humanized antibody activity

[0180] 4.1 Construction and preparation of humanized antibodies

[0181] The amino acid sequences of the heavy and light chain variable regions of 589E2D4 were analyzed, and three antigen complementarity-determining regions (CDRs) and four frame regions (FRs) were identified according to Kabat rules. The amino acid sequences of the heavy chain CDRs were HCDR1: DYEMH (SEQ ID NO: 8), HCDR2: AIDPETGGTDYNQKFKG (SEQ ID NO: 9), and HCDR3: SYDYGVDWYFDV (SEQ ID NO: 10), while the amino acid sequences of the light chain CDRs were LCDR1: RSSQSLVHSNGNTYLH (SEQ ID NO: 11), LCDR2: KVSNRFS (SEQ ID NO: 12), and LCDR3: SQSTHVPYT (SEQ ID NO: 13).

[0182] Homology comparison between NCBI IgBlast and human IgG germline sequences was performed, and IGHV1-3*01 was selected as the heavy chain CDR transplantation template, while IGKV2-28*01 was selected as the light chain CDR transplantation template. The CDR region of the 589E2D4 antibody was transplanted onto the selected humanized templates, replacing the CDR region of the human template. The heavy chain variable region was then recombined with the human IgG1 constant region, and the light chain variable region was recombined with the human kappa chain constant region. Simultaneously, based on the three-dimensional structure of this antibody, reverse mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the antibody's VL and VH regions, resulting in multiple humanized antibodies. Affinity screening was then used to determine the heavy chain variable region sequence (SEQ ID NO: 14) and light chain variable region sequence (SEQ ID NO: 15) of the humanized antibody 589-81. The humanized antibody 589-81 was prepared according to the method described in Example 3.2 for chimeric antibody construction.

[0183] 4.2 Humanized antibody cell-binding activity

[0184] The binding activity of humanized antibody 589-81 with HumanIL-2Rbeta / IL-2R gamma(Luc)HEK293 Reporter Cell was determined according to the "Experimental Method for Cell Binding Activity" in Example 2.2.

[0185] Experimental results are as follows Figure 5 As shown, the humanized antibody 589-81 exhibits stronger cell-binding activity than the positive control antibody MAB05.

[0186] 4.3 Assay of the binding kinetics of humanized monoclonal antibody to human CD122

[0187] Antibodies were replenished using a chip covalently coupled with Protein A / G. The relevant operating parameters were as follows: antibody concentration 2 μg / mL, contact time 75 s, flow rate 10 μL / min, and regeneration contact time 30 s. The hCD122-His antigen was diluted with HBS-EP pH 7.4 buffer (prepared as in Example 1), with a maximum concentration of 100 nM. It was serially diluted 2-fold to 0.78 nM. Replicas and a 0 concentration point were set. 6 M guanidine hydrochloride solution was used as the regeneration buffer. The sample was injected into the Biacore 8K chip with the following parameters: binding time 180 s, dissociation time 900 s, flow rate 30 μL / min, and regeneration contact time 30 s. After the run, the data were analyzed using Biacore 8K Evaluation Software according to the "1:1 binding kinetics model" to obtain the binding kinetic parameters of each antibody to hCD122-His.

[0188] The binding constant (ka), dissociation constant (kd), and equilibrium dissociation constant (KD) of humanized antibody 589-81 and positive control antibody MAB05 to hCD122-His are shown in Table 1. The affinity of 589-81 for hCD122-His is significantly higher than that of MAB05.

[0189] Table 1: Binding kinetic parameters of each humanized antibody to hCD122-His

[0190] Sample ka (1 / Ms) kd (1 / s) KD (M) 589-81 4.17E+06 2.07E-03 4.96E-10 MAB05 3.20E+05 2.10E-03 6.55E-09

[0191] 4.4 Cell blocking activity of humanized antibodies

[0192] Referring to the "Cellular Blocking Activity Assay" in Example 2, the blocking activity of humanized antibody 589-81 against the binding of Human IL-2Rbeta / IL-2R gamma (Luc) HEK293 Reporter Cell to hIL-15 was determined. The experimental results are as follows: Figure 6 As shown, the humanized antibody 589-81 exhibits stronger cell-blocking activity than the positive control antibody MAB05.

[0193] 4.5 Humanized antibodies inhibit IL-15-activated cells

[0194] Human IL-2R beta / IL-2R gamma(Luc)HEK293 Reporter Cells were seeded into white 96-well plates at a concentration of 5e4 per well. The antibody was serially diluted with complete culture medium and added to the cells. The plates were incubated at 37°C for 1 hour. Then, hIL-15 was added to each well to a final concentration of 10 ng / mL, and the plates were incubated at 37°C for 6 hours. The cells were then removed and allowed to return to room temperature. 100 μL of Bio-Glo mixture (already at room temperature) was added to each well, and the plates were incubated at room temperature in the dark for 10 minutes. The results were then analyzed. (Results are shown below.) Figure 7 As shown, 589-81 showed stronger inhibition of IL15-activated cell activity than the positive control antibody MAB05.

[0195] 4.6 Humanized antibodies inhibit IL-15-induced M07e cell proliferation

[0196] M07e acute megakaryocytic leukemia cell line was seeded into white 96-well plates at a concentration of 5e4 per well. Antibody was serially diluted with GM-CSF-free complete medium and added to the cells. hIL-15 protein was added to achieve a final concentration of 50 ng / mL. The plates were then incubated at 37°C for 72 h. Afterward, the 96-well plates were removed, and 10% (20 μL) of CCK8 reagent was added to each well. The mixture was thoroughly combined, and the plates were incubated at 37°C for another 2-3 h. The OD450 was then measured after thorough mixing. The experimental results are as follows: Figure 8 As shown, 589-81 exhibits significantly stronger inhibitory activity against M07e proliferation than the positive control antibody MAB05, suggesting its potential for treating diseases related to the IL-15 signaling pathway, such as leukemia.

[0197] 4.7 Humanized antibodies inhibit IFNγ secretion by PBMC cells

[0198] First, PBMC cells were centrifuged at 600g for 10 minutes, the supernatant was discarded, and the cells were washed once with complete culture medium. After centrifugation, the cells were resuspended in complete culture medium, counted, and seeded at a concentration of 5e5 per well. The antibody was serially diluted and added to the cell wells, along with hIL-15 to a final concentration of 100 ng / mL. After thorough mixing, the cells were incubated at 37°C for 72 h. Subsequently, the cells were removed, mixed thoroughly, centrifuged, and the supernatant was collected for assay according to the IFNγ kit instructions. The experimental results are as follows: Figure 9 As shown, the humanized antibody 589-81 significantly inhibited the secretion of IFNγ by PBMC cells compared to the positive control.

[0199] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this application should still be covered by the claims of this application.

Claims

1. A CD122 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region, said heavy chain variable region comprising HCDR1-3 as shown in SEQ ID NO: 8-10.

2. The CD122 antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO:

14.

3. The CD122 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The CD122 antibody or its antigen-binding fragment further includes a light chain variable region, which includes LCDR1-3 as shown in SEQ ID NO: 11-13.

4. The CD122 antibody or its antigen-binding fragment according to claim 3, characterized in that, The light chain variable region includes an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO:

15.

5. The CD122 antibody or its antigen-binding fragment according to claim 3, characterized in that, The CD122 antibody or its antigen-binding fragment is selected from any of the following: The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 5; Alternatively, the heavy chain variable region may include an amino acid sequence as shown in SEQ ID NO: 14, and the light chain variable region may include an amino acid sequence as shown in SEQ ID NO:

15.

6. The CD122 antibody or its antigen-binding fragment according to claim 3, wherein the heavy chain variable region and / or light chain variable region further comprises amino acid mutations; Preferably, the amino acid mutation is located in the FR framework region; Preferably, the heavy chain variable region is selected from amino acid sequences that have at least 99%, 95%, 90%, or 85% identity with and have the same function as SEQ ID NO: 3 and 14; and / or, the light chain variable region is selected from amino acid sequences that have at least 99%, 95%, 90%, or 85% identity with and have the same function as SEQ ID NO: 5 and 15. Preferably, the antibody further includes a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is the heavy chain constant region of a human or mouse antibody, and the light chain constant region is the light chain constant region of a human or mouse antibody. Preferably, the heavy chain constant region is the IgG1 heavy chain constant region, and the light chain constant region is the Kappa light chain constant region; Preferably, the constant region includes amino acid mutations; Preferably, the heavy chain sequence of the CD122 antibody or its antigen-binding fragment is shown in SEQ ID NO:16, and the light chain sequence of the CD122 antibody or its antigen-binding fragment is shown in SEQ ID NO:

17.

7. The CD122 antibody or its antigen-binding fragment according to claims 1 to 6, characterized in that, The antibody is selected from polyclonal antibodies, monoclonal antibodies, single-chain antibodies, antigen-binding domains, bispecific antibodies, multispecific antibodies, or antigen-binding portions of chimeric antigen receptors; And / or, the antigen-binding fragment is selected from scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv; And / or, the antibody is selected from murine antibodies, chimeric antibodies, and humanized antibodies.

8. A polynucleotide encoding a CD122 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7.

9. A vector comprising the polynucleotide as described in claim 8.

10. A host cell containing the vector as described in claim 9, or the host cell having the polynucleotide as described in claim 8 integrated into its genome.

11. An antibody-drug conjugate comprising a therapeutic agent, a linker, and a CD122 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7.

12. A pharmaceutical composition comprising an effective amount of the CD122 antibody or its antigen-binding fragment as described in any one of claims 1 to 7, or the antibody-drug conjugate as described in claim 11.

13. A kit for detecting CD122, the kit comprising an effective amount of the CD122 antibody or its antigen-binding fragment as described in any one of claims 1 to 7 and a detection reagent.

14. Use of the CD122 antibody or its antigen-binding fragment as described in any one of claims 1 to 7, or the polynucleotide as described in claim 8, or the vector as described in claim 9, or the host cell as described in claim 10, or the antibody-drug conjugate as described in claim 11, or the pharmaceutical composition as described in claim 12, wherein the use is selected from at least one of the following: 1) To prepare products for the prevention and / or treatment of diseases related to IL-15, and / or IL-2, and / or CD122; 2) To prepare products for the prevention and / or treatment of immune system diseases, inflammatory diseases, and tumors; Preferably, the diseases associated with IL-15, and / or IL-2, and / or CD122 are selected from vitiligo, alopecia areata, type 1 diabetes, atopic dermatitis, ulcerative colitis, systemic lupus erythematosus, eczema, psoriasis, celiac disease, graft-versus-host disease, and leukemia; Preferably, the tumor is selected from solid tumors or non-solid tumors; Preferably, the product is selected from pharmaceuticals and reagents; Preferably, the product has one or more of the following effects: 1) Inhibits IL-15 / IL-2 activation of cells; 2) Inhibits IL-15 / IL-2-mediated tumor cell proliferation; 3) Inhibits the secretion of pro-inflammatory factors by immune cells mediated by IL-15 / IL-2; Preferably, the cells, tumor cells, and immune cells express CD122.

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