PRLR antigen-binding protein, its production method and use

Abstract

Provided are a PRLR antigen-binding protein, a method for producing the same, and uses thereof. By hybridoma technology, a PRLR antigen-binding protein having good affinity and binding activity for human PRLR-ECD and capable of effectively inhibiting the activation of PRLR has been obtained. The PRLR antigen-binding protein can be used for treating various PRLR-positive diseases such as tumors and alopecia.

Description

[Technical Field]

[0001] The present invention belongs to the field of biomedicine, and specifically relates to PRLR antigen-binding proteins, their production methods and uses. [Background technology]

[0002] Prolactin (PRL), also known as lactogenic hormone or lactotropic hormone, is an anterior pituitary peptide hormone required for successful reproduction. Its gene family includes the prolactin receptor (PRLR), growth hormone receptor (GHR), and placental lactogen receptor (PLR). The PRLR is a cell membrane receptor expressed in the mammary gland, ovary, pituitary gland, heart, lung, thymus, spleen, liver, pancreas, kidney, adrenal gland, uterus, skeletal muscle, skin, and central nervous system. It is involved in various physiological functions, including cell proliferation, differentiation, lactation, and reproduction.

[0003] Mammalian PRLs are known to have at least two receptor forms: a long form and a short form. The structural diversity of PRLRs is primarily due to mutations in the intracellular domain. PRLRs are transmembrane proteins with only one transmembrane domain. They consist of three domains: an extramembrane domain, a transmembrane domain, and an intracellular domain. These domains share the structural characteristics of the cytokine receptor superfamily, and each domain is composed of two domains: fibronectin type III 1 and fibronectin type III 2. The PRLR structures of mammals and birds differ in that mammals have only one extracellular ligand-binding domain, while birds have two repeating units of the ligand-binding domain. The genomic structure of the human PRLR gene has already been elucidated (Hu, Z.-Z. et al., J. Clin. Endocr. Metab. 84:1153-1156, 1999). The 5'-untranslated region of the PRLR gene contains two first exons, E13, which are the human counterparts of rat and mouse E13, and a novel human first exon, designated E1N. The 5'-untranslated region also contains one universal noncoding exon, 2, and part of exon 3, the latter of which contains the translation initiation codon. The E13 and E1N exons are less than 800 base pairs long. These two exons are expressed in human mammary tissue, breast cancer cells, gonads, and liver. In summary, transcripts containing E13 are expressed in most tissues. The PRLR gene product is encoded by exons 3 to 10, of which exon 10 encodes the majority of the intracellular region. Transcription of the E13 and E1N exons is initiated from either the PIII or PN promoters, respectively. The PIII promoter contains Sp1 and C / EBP elements that are identical to those in rodent promoters and share 81% homology with the -480 / -106 region of the rat and mouse gene. The PN promoter contains putative binding sites for ETS family proteins and half-sites for nuclear receptors.

[0004] Activation of PRL-R by PRL-R binding to its receptor and formation of a trimer is a prerequisite for various biological actions. Following activation, PRL-R phosphorylates JAK2, inducing distal tyrosine phosphorylation within the receptor cell, activating STAT proteins, which then translocate to the nucleus and activate target gene transcription, resulting in biological effects. This is a fundamental pattern by which PRL, a mediator of the neuroendocrine-immune network, regulates immune function. Furthermore, after binding to its receptor, PRL activates the PI3K and MAPK signaling pathways, increasing cyclin D1 expression and promoting breast cancer cell proliferation.

[0005] PRLR is highly expressed in various endocrine-related tumor cells, including breast, prostate, and ovarian cancers. It is associated with the development and progression of these tumors, and elevated circulating levels of its ligand, prolactin (PRL), are a high-risk factor for breast cancer development and a negatively correlated indicator of prognosis in breast cancer patients. Recent studies have demonstrated that PRLR activation is associated with alopecia, and elevated circulating prolactin levels are closely related to alopecia. Therefore, PRLR is a potential drug target, and effective inhibition of PRLR activation can treat various related diseases. Patent CN200780030284.7 discloses a PRLR-specific antibody, a pharmaceutical composition containing this antibody, a kit containing the pharmaceutical composition, and a method for preventing and treating cancer. Patent CN201480055822.8 also discloses an antibody that binds to the prolactin receptor (PRLR) and a method for using the same. The antibody binds to human PRLR with high affinity, blocks prolactin-mediated signal transduction, and can be used to treat various cancers and other PRLR-related conditions.

[0006] However, there are few antibodies against PRLR in the prior art. Therefore, there is an urgent need to provide a novel PRLR-specific antibody with high affinity that can bind to PRLR and inhibit PRLR activation, and can be used to treat various PRLR-related diseases such as tumors and alopecia. Summary of the Invention [Problem to be solved by the invention]

[0007] In response to the above-mentioned problems, the present invention provides novel PRLR antigen-binding proteins, as well as methods for producing and using the same. Using hybridoma technology, the present invention obtains a series of novel PRLR antigen-binding proteins with biological functions, which have good affinity and binding activity for human PRLR-ECD and inhibit PRLR activation. The PRLR antigen-binding proteins described in the present invention can be used to treat various PRLR-related diseases, such as tumors and alopecia, providing a new approach to the treatment of PRLR-related diseases. [Means for solving the problem]

[0008] The technical solutions of the present invention to achieve the above object of the invention are as follows: In one aspect, the present invention provides a PRLR antigen-binding protein.

[0009] Specifically, the PRLR antigen-binding protein has the following complementarity determining regions: (1) Heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof (2) Heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof (3) Heavy chain complementarity-determining region 3 (HCDR3) containing the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof (4) Light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence shown in SEQ ID NO: 15 or a variant thereof (5) Light chain complementarity-determining region 2 (LCDR2) comprising the amino acid sequence shown in SEQ ID NO: 16 or a variant thereof (6) a light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 17 or a variant sequence thereof; Preferably, said variant sequence is a CDR sequence which has one or more amino acid substitutions, deletions or additions compared to the CDR from which it is derived, said substitutions being conservative substitutions.

[0010] More specifically, the PRLR antigen-binding protein is (1) a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 2 and / or a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 4 or (2) a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VH of (1), and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VL of (1). or (3) a VH having one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared to the VH of (1), and / or a VL having one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared to the VL of (1), wherein the substitutions are conservative substitutions.

[0011] More specifically, the PRLR antigen-binding protein further comprises a human IgG1 constant region and a human kappa (κ) chain constant region, the amino acid sequence of the human IgG1 constant region being as set forth in SEQ ID NO:6, and the amino acid sequence of the human kappa (κ) chain constant region being as set forth in SEQ ID NO:7.

[0012] More specifically, the VH of the PRLR antigen-binding protein is linked to a human IgG1 constant region to form a heavy chain, and the VL of the PRLR antigen-binding protein is linked to a human κ (kappa) chain constant region to form a light chain.

[0013] More specifically, the PRLR antigen-binding protein is (1) a heavy chain HC comprising the amino acid sequence shown in SEQ ID NO: 8 and / or a light chain LC comprising the amino acid sequence shown in SEQ ID NO: 9 or (2) heavy and light chains, wherein the heavy chains have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and / or the light chains have at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the heavy and light chains of (1).

[0014] More specifically, the above PRLR antigen-binding proteins include chimeric antibodies, humanized antibodies, or fully human antibodies.

[0015] More specifically, the PRLR antigen-binding proteins include full-length antibodies, Fab, Fab', F(ab')2, Fv, scFv, di-scFv, bispecific antibodies, multispecific antibodies, heavy chain antibodies and / or single domain antibodies, or monoclonal and / or polyclonal antibodies produced by the above antibodies.

[0016] In another aspect, the present invention provides a humanized PRLR antigen binding protein, the humanized PRLR antigen binding protein comprising: (1) a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 18 and / or a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 19 or (2) a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VH of (1), and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VL of (1). or (3) a VH having one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared to the VH of (1), and / or a VL having one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared to the VL of (1), wherein the substitutions are conservative substitutions.

[0017] Specifically, the humanized PRLR antigen-binding protein comprises: (1) a heavy chain HC comprising the amino acid sequence shown in SEQ ID NO: 20 and / or a light chain LC comprising the amino acid sequence shown in SEQ ID NO: 21 or (2) heavy and light chains, wherein the heavy chains have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and / or the light chains have at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the heavy and light chains of (1).

[0018] In another aspect, the present invention provides a series of nucleic acid molecules encoding the PRLR antigen-binding protein or humanized PRLR antigen-binding protein.

[0019] Specifically, the nucleic acid molecules include one or more nucleic acid molecules that are codon-optimized.

[0020] In another aspect, the present invention provides a series of vectors comprising one or more of the nucleic acid molecules described herein.

[0021] Specifically, said vectors include, but are not limited to, plasmids, viruses and phages.

[0022] In another aspect, the present invention provides a series of host cells comprising the above nucleic acid molecule or the above vector.

[0023] Specifically, the host cells include, but are not limited to, microbial, plant, or animal cells, and the vectors described in the present invention may be introduced into the host cells by methods known to those skilled in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, etc.

[0024] In another aspect, the present invention provides a chimeric antigen receptor comprising the above-mentioned PRLR antigen-binding protein or humanized PRLR antigen-binding protein.

[0025] In another aspect, the present invention provides an immune cell comprising the above-mentioned chimeric antigen receptor.

[0026] In another aspect, the present invention provides an antigen-binding protein derivative comprising the above-mentioned PRLR antigen-binding protein or humanized PRLR antigen-binding protein and a detectable label molecule.

[0027] Specifically, the detectable label molecule is an enzyme (eg, horseradish peroxidase), a radioisotope, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.

[0028] In another aspect, the present invention provides a multispecific antibody comprising the above-mentioned PRLR antigen-binding protein or humanized PRLR antigen-binding protein and another antibody or a fragment thereof, or an antibody mimetic.

[0029] In particular, said multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

[0030] In another aspect, the present invention provides an antibody-drug conjugate comprising an antibody portion and a binding portion, wherein the antibody portion comprises the PRLR antigen-binding protein or humanized PRLR antigen-binding protein described above, and the binding portion comprises, but is not limited to, a detectable marker, a drug, a toxin, a cytokine, a radioisotope, an enzyme, or a combination thereof, and the antibody portion and the binding portion are linked by a chemical bond or a linker.

[0031] In another aspect, the present invention provides pharmaceutical compositions comprising the above-mentioned PRLR antigen-binding protein, humanized PRLR antigen-binding protein, nucleic acid molecule, vector, host cell, chimeric antigen receptor, immune cell, antigen-binding protein derivative, multispecific antibody and / or antibody-drug conjugate.

[0032] In particular, the pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier.

[0033] More specifically, the pharmaceutically acceptable carrier includes, but is not limited to, a diluent, an excipient, a filler, a wetting agent, a disintegrant, a flavoring agent, and a binder.

[0034] Specifically, the pharmaceutical composition further comprises a co-therapeutic agent, which includes, but is not limited to, a chemotherapeutic agent, a radiotherapeutic agent, an immunosuppressant, and a cytotoxic drug.

[0035] In another aspect, the present invention provides a method for producing the above-mentioned PRLR antigen-binding protein or humanized PRLR antigen-binding protein, said method comprising culturing the above-mentioned host cell whereby the antigen-binding protein is expressed.

[0036] In another aspect, the present invention provides use of the PRLR antigen-binding protein, humanized PRLR antigen-binding protein, nucleic acid molecule, vector, host cell, chimeric antigen receptor, immune cell, antigen-binding protein derivative, multispecific antibody, antibody-drug conjugate and / or pharmaceutical composition in the manufacture of a PRLR blocking agent, kit and / or medical device.

[0037] Specifically, the PRLR blocker, kit and / or device is mainly used for diseases in which the expression level of PRLR increases.

[0038] In another aspect, the present invention provides use of the PRLR antigen-binding protein, humanized PRLR antigen-binding protein, nucleic acid molecule, vector, host cell, chimeric antigen receptor, immune cell, antigen-binding protein derivative, multispecific antibody, antibody-drug conjugate and / or pharmaceutical composition in the manufacture of a drug, kit and / or administration device for preventing and / or treating a PRLR-positive disease.

[0039] Specifically, the PRLR-positive disease is a tumor or alopecia.

[0040] More specifically, the tumors include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, lung cancer, skin cancer, pancreatic cancer, kidney cancer, stomach cancer, and the like.

[0041] In another aspect, the present invention provides use of the PRLR antigen-binding protein, humanized PRLR antigen-binding protein and / or antigen-binding protein derivative in the manufacture of a PRLR detection reagent or kit.

[0042] In another aspect, the present invention provides a PRLR detection method for qualitatively or quantitatively analyzing and detecting PRLR using the above-mentioned PRLR antigen-binding protein or humanized PRLR antigen-binding protein, wherein the detection method is used for purposes other than the diagnosis or treatment of disease.

[0043] In another aspect, the present invention provides a method for treating a PRLR-positive associated disease, comprising administering an effective amount of the above-mentioned PRLR antigen-binding protein, humanized PRLR antigen-binding protein, immune cell, antigen-binding protein derivative, multispecific antibody, antibody-drug conjugate and / or pharmaceutical composition to a subject in need thereof.

[0044] Specifically, the PRLR-positive disease is a tumor or alopecia.

[0045] More specifically, the tumors include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, lung cancer, skin cancer, pancreatic cancer, kidney cancer, stomach cancer, and the like.

[0046] In another aspect, the present invention provides a kit comprising the PRLR antigen-binding protein, humanized PRLR antigen-binding protein, nucleic acid molecule, vector, host cell, chimeric antigen receptor, immune cell, antigen-binding protein derivative, multispecific antibody, antibody-drug conjugate and / or pharmaceutical composition, and, optionally, instructions.

[0047] In another aspect, the present invention provides an administration device comprising: (1) an injection module used to administer the pharmaceutical composition to a subject in need thereof; and (2) optionally, a drug efficacy monitoring module. [Effects of the Invention]

[0048] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a novel PRLR antigen-binding protein, which is obtained using hybridoma technology and has good affinity and binding activity for human PRLR-ECD, and can effectively inhibit PRLR activation.

[0049] (2) The PRLR antigen-binding protein described in the present invention can be used to treat various PRLR-positive diseases such as tumors and alopecia, providing a new idea for the treatment of PRLR-positive diseases. [Brief explanation of the drawings]

[0050] [Figure 1] 1 shows the results of measuring the binding affinity of mouse monoclonal antibody SPGA02-260 to human PRLR-ECD. [Figure 2] 1 shows the results of measuring the binding affinity of mouse monoclonal antibody SPGA02-260 to T47D cells highly expressing human PRLR. [Figure 3] This shows the results of measuring the inhibition of PRL signal transduction (STAT5 phosphorylation) by the mouse monoclonal antibody SPGA02-260. [Figure 4] This shows the results of measuring the inhibition of PRL signal transduction (ERK1 / 2 phosphorylation) by the mouse monoclonal antibody SPGA02-260. [Figure 5] This shows the results of measuring endocytosis caused by the binding of mouse monoclonal antibody SPGA02-260 to T47D cells. [Figure 6] 1 shows the results of measuring the binding affinity of chimeric antibody SPGA02-ch260 to human PRLR-ECD. [Figure 7] 1 shows the results of measuring the binding affinity of chimeric antibody SPGA02-ch260 to T47D cells that highly express human PRLR. [Figure 8] 1 shows the results of measuring the binding affinity of chimeric antibody SPGA02-ch260 to mouse PRLR-ECD. [Figure 9]This shows the results of measuring the binding ability of SPGA02-260 to different binding regions of the target antigen PRLR-ECD. [Figure 10] 1 shows the results of measuring the binding of SPGA02-260 to the target antigen PRLR-ECD polypeptide. [Figure 11] This shows the results of measuring the binding of humanized monoclonal antibody SPGA02-hu260 to mouse PRLR. [Figure 12] This is the result of measuring the inhibition of PRL-induced signal transduction (STAT5 phosphorylation) by the humanized monoclonal antibody SPGA02-hu260. [Figure 13] This is the result of measuring the inhibition of PRL-induced signaling (ERK1 / 2 phosphorylation) by the humanized monoclonal antibody SPGA02-hu260. [Figure 14] This is a measurement of the inhibition of GH1-induced signaling (STAT5 phosphorylation) by the humanized monoclonal antibody SPGA02-hu260. [Figure 15] This is the result of measuring the inhibition of GH1-induced signaling (ERK1 / 2 phosphorylation) by the humanized monoclonal antibody SPGA02-hu260. [Figure 16] 1 shows the results of measuring the inhibition of the humanized monoclonal antibody SPGA02-hu260 on the proliferation of BAF3-huPRLR cells. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention will be described in more detail below using specific examples. The following examples are merely for the purpose of illustrating the present invention and are not intended to limit the present invention. Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions for those experimental methods for which specific conditions are not specified in the examples. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available products.

[0052] (Term definition) To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless expressly defined herein, all other technical and scientific terms used herein have the ordinary meanings understood by those skilled in the art.

[0053] The three-letter and one-letter codes of amino acids used in the present invention refer to J. Biol. Chem, 243, p. 3558 (1968, IUPAC-IUB Commission).

[0054] The term "antigen-binding protein" as used herein generally refers to a protein comprising an antigen-binding portion and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to an antigen. Typically, the protein may comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. The VH and VL regions are further divided into hypervariable regions called complementarity-determining regions (CDRs), which are dispersed within more conserved regions called framework regions (FR or FWR). The variable regions of the heavy and light chains contain binding domains that interact with antigens. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', Fv fragments, F(ab')2, scFv, dis-scFv, and / or dAb), immune complexes, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody mimetics, chimeric antigen receptors, or fusion proteins, as long as they exhibit a predetermined antigen-binding activity.

[0055] The term "antibody" as used herein refers to an immunoglobulin, a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The antigenicity of immunoglobulins varies due to differences in the amino acid composition and sequence of the heavy chain constant regions. Therefore, immunoglobulins may be classified into five types, also known as immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig subclasses of the same type may be classified into different subclasses based on the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG may be classified into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ or λ chains based on the constant region. Each of the five types of Ig may have either κ or λ chains.

[0056] The sequences of approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains are largely different and constitute the variable region (Fv region), while the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region. The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). The light chain variable region (VL or LCVR) and the heavy chain variable region (VH or HCVR) each consist of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

[0057] The term "complementarity-determining region (CDR)" refers to one of the six hypervariable regions in the variable domain of an antibody that are primarily responsible for binding to an antigen. Generally, there are three CDRs (HCDR1, HCDR2, and HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region. CDRs may be determined based on various numbering systems known in the art, such as the Kabat, Chothia, AbM, or IMGT numbering systems. In the present invention, the "Kabat numbering convention" (see Kabat et al. (1991)) is used to determine the amino acid sequence boundaries of CDRs.

[0058] The term "framework region" or "FR" residues refers to amino acid residues of antibody variable regions other than the CDR residues as defined above.

[0059] The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous antibody population in which, except for possible variant antibodies, the individual antibodies comprising the population are identical and / or bind to the same epitope. Unlike polyclonal antibody preparations, which generally include different antibodies directed against different determinants, a monoclonal antibody preparation has each monoclonal antibody directed against a single determinant on an antigen. Thus, "monoclonal" refers to the character of an antibody obtained from a substantially homogeneous antibody population and is not intended to refer to production of the antibody by any particular method. Monoclonal antibodies according to the present invention may be produced by a variety of techniques known to those of skill in the art, including, but not limited to, hybridoma, recombinant DNA, phage display, and transgenic methods.

[0060] The term "humanized monoclonal antibody" refers to an antibody produced by grafting mouse CDR sequences onto the variable region framework of a human antibody, i.e., onto a different type of human germline antibody framework sequence. This overcomes the xenogeneic response that can occur when chimeric antibodies contain a large amount of mouse protein components. To avoid reduced immunogenicity and reduced activity, minimal back mutations may be made to the variable region framework sequences of the human antibody to maintain activity. To produce a humanized antibody, mouse CDR regions may be inserted into human framework sequences using methods known in the art (see U.S. Patent No. 5,225,539 by Winter; U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 by Queen et al.; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals may be used that do not produce endogenous immunoglobulins and are capable of producing a fully human antibody repertoire after immunization (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; Duchosal et al., 1992, Nature 355:258; Lonberg et al. (1994) Nature 368(6474):856-859; International Patent Publication WO 02 / 43478). Other methods for humanizing antibodies include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0061] The terms "specific binding" and "selective binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction is determined by the dissociation equilibrium constant (K D ) As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which represents the binding affinity between the antibody and the antigen. The smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind to antigens at a concentration of about 10 -8 Less than m, e.g., about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Affinity (K D ) to join them.

[0062] The term "identity" generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical to those in a second reference polypeptide sequence or portion thereof, after aligning the sequences, optionally introducing gaps (GAPS), to achieve the maximum percent sequence identity; however, any conservative substitutions are not taken into account when determining sequence identity. Alignment to determine percent amino acid / nucleotide sequence identity can be achieved using various methods known in the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for measurement and alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. Percent identity may be measured over the entire length of the determined polypeptide / polynucleotide sequence, or over a shorter length, for example, the length of a fragment obtained from the determined long polypeptide / polynucleotide sequence. It should be understood that the length of any fragment obtained from a sequence shown in a table, figure, or sequence listing herein may be the length used to determine percent identity. A sequence with "percent identity" retains the significant biological activity (such as antibody binding specificity) of the sequence to which it is compared or derived. A sequence with one or more amino acid substitutions, deletions, or additions, or any combination thereof, retains the significant biological activity (such as antibody binding specificity) of the sequence to which it is compared or derived. Nucleotide sequences with "percent identity" or that differ by no more than 3, 6, 15, 30, or 45 nucleotides can perform a similar function to the nucleotide sequence to which it is compared or derived, e.g., the expressed proteins can both specifically bind to the same antigen or molecule.

[0063] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis, etc. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, e.g., substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties (such as the ability to form covalent or hydrogen bonds)). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable to replace a corresponding amino acid residue with another amino acid residue from the same family that has the same side chain. Methods for identifying conservative amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); Burks et al., Proc. Natl. Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0064] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector may be introduced into a host cell by transformation, transduction, or transfection so that the genetic material element carried therein is expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), phages (such as lambda phage or M13 phage), and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). Vectors may contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, reporter genes, and may further contain an origin of replication site.

[0065] The term "host cell" generally refers to an individual cell, cell line, or cell culture that can contain or has contained a plasmid or vector containing a nucleic acid molecule described herein, or that can express an antibody or antigen-binding fragment thereof described herein. The cell may include the progeny of a single host cell. Due to natural, accidental, or deliberate mutation, the progeny may not necessarily be completely morphologically or genomicly identical to the original parent cell, but need only be capable of expressing the antibody or antigen-binding fragment thereof described herein. The cell may be obtained by transfecting a cell in vitro with a vector described herein. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., yeast cell, COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell). Optionally, the cell may be a mammalian cell. For example, the mammalian cell may be a CHOK1 cell.

[0066] The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, as is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption retarders, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as, but not limited to, parahydroxybenzoates, chlorobutanol, phenol, sorbic acid, and the like. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and analogs thereof. Absorption retardants include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (such as phosphate-buffered saline), alcohols, and polyols (such as glycerin). Preservatives include various antibacterial and antifungal agents, such as, but not limited to, thimerosal, 2-phenoxyethanol, parahydroxybenzoates, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the usual meaning understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in a drug, and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid or glycine), proteins (such as dried whey, albumin, or casein), or their hydrolyzates (such as lactalbumin hydrolysate), and the like.

[0067] The term "pharmaceutical composition" generally refers to a formulation in which the biological activity of the active ingredient is present in a form that allows it to be effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. The composition is sterile.

[0068] The term "subject" refers to a mammal, e.g., a primate mammal, e.g., a non-human primate mammal, or a human. In some embodiments, the subject (e.g., a human) has or is at risk of having a disease, such as a tumor or alopecia (including, but not limited to, breast cancer, prostate cancer, ovarian cancer, lung cancer, skin cancer, pancreatic cancer, kidney cancer, stomach cancer, etc.).

[0069] The term "effective amount" refers to an amount sufficient to achieve a desired effect, or a portion thereof. For example, an effective amount for preventing a disease (e.g., PRLR-positive associated) refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., PRLR-positive associated disease), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient suffering from the disease. Determining this effective amount is within the capabilities of those skilled in the art. For example, an effective amount for treatment is determined based on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition (e.g., age, weight, sex), the mode of drug administration, other treatments being administered at the same time, etc.

[0070] The term "chimeric antigen receptor" refers to chimeric antigen receptor T cells, which combine the antigen-binding site of an antibody that recognizes a specific antigen in vitro with the intracellular portion of the CD3-δ chain or FcεRIγ chain to obtain a chimeric protein. Patients' T cells are then transfected to express the chimeric antigen receptor by transduction, allowing the patient's T cells to be "recoded" and subsequently generate large quantities of specific CAR-T cells. When the recoded chimeric antigen receptor T cells are introduced into the patient's body, the chimeric antigen receptor acts like a GPS to specifically track and recognize T cells and guide them to kill them. Most chimeric antigen receptors consist of an extracellular antigen-binding region (composed of light and heavy chains derived from monoclonal antibodies, connected by a flexible hinge region to form a single-chain antibody), a transmembrane domain, and an intracellular signaling region. CAR structures are obtained by in vitro genetic recombination of an scFv that recognizes the relevant antigen and an intracellular signaling domain called an "immunoreceptor tyrosine-based activation motif."

[0071] The term "immune cell" includes cells of hematopoietic origin that play a role in the immune response, such as lymphocytes (e.g., B cells, T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, granulocytes).

[0072] (abbreviation) CDRs are complementarity determining regions in immunoglobulin variable regions. VH is the antibody heavy chain variable region. VL is the antibody light chain variable region. HC is the antibody heavy chain. LC is the antibody light chain. IgG is immunoglobulin G. AbM is the AbM CDR definition system based on Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), which integrates parts of both the Kabat and Chothia definitions. Kabat is an immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). Chothia is an immunoglobulin numbering system proposed by Chothia et al., which is a classical convention for identifying the boundaries of CDR regions based on the location of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). IMGT is a numbering system based on The international ImMunoGeneTics information system® (IMGT) devised by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. mAb is a monoclonal antibody. EC 50 is the concentration at which 50% efficacy or binding is observed. I C 50 is the concentration at which 50% inhibition is observed. ELISA is an enzyme-linked immunosorbent assay. PCR is the polymerase chain reaction. HRP is horseradish peroxidase. PRLR is the prolactin receptor. hFc is the human IgG antibody Fc region. K D is the dissociation equilibrium constant. HCDR1 is the complementarity determining region 1 in the immunoglobulin heavy chain variable region. HCDR2 is the complementarity determining region 2 in the immunoglobulin heavy chain variable region. HCDR3 is the complementarity determining region 3 in the immunoglobulin heavy chain variable region. LCDR1 is the complementarity determining region 1 in the immunoglobulin light chain variable region. LCDR2 is the complementarity determining region 2 in the immunoglobulin light chain variable region. LCDR3 is the complementarity determining region 3 in the immunoglobulin light chain variable region.

[0073] "Detailed Description of the Invention" (antigen-binding protein) The present invention provides a series of PRLR antigen-binding proteins, which are produced by mouse immunization, molecular biology and antibody engineering techniques.

[0074] In some embodiments, the antigen binding proteins provided by the present invention comprise the following complementarity determining regions: (1) Heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof (2) Heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof (3) Heavy chain complementarity-determining region 3 (HCDR3) containing the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof (4) Light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence shown in SEQ ID NO: 15 or a variant thereof (5) Light chain complementarity-determining region 2 (LCDR2) comprising the amino acid sequence shown in SEQ ID NO: 16 or a variant thereof (6) a light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 17 or a variant sequence thereof; Preferably, said variant sequence is a CDR sequence which has one or more amino acid substitutions, deletions or additions compared to the CDR from which it is derived, said substitutions being conservative substitutions.

[0075] In some embodiments, the antigen binding proteins provided by the present invention comprise the following heavy chain variable region, VH, and light chain variable region, VL: (1) a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 2 and / or a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 4 or (2) a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VH of (1), and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VL of (1). or (3) a VH having one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared to the VH of (1), and / or a VL having one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared to the VL of (1), wherein the substitutions are conservative substitutions.

[0076] In some embodiments, the antigen binding proteins provided by the present invention comprise: The following heavy chain HC and light chain LC: (1) a heavy chain HC comprising the amino acid sequence shown in SEQ ID NO: 8 and / or a light chain LC comprising the amino acid sequence shown in SEQ ID NO: 9 or (2) heavy and light chains, wherein the heavy chains have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and / or the light chains have at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the heavy and light chains of (1).

[0077] In some specific embodiments, the PRLR antigen binding proteins produced by the present invention comprise a combination of heavy and light chains as set forth in Table 1 below. [Table 1] Table 1: Sequence characteristics of PRLR antigen-binding proteins JPEG0007827890000001.jpg117147

[0078] (Antibody derivatives and multispecific antibodies) The antigen-binding proteins provided by the present invention, as described above, include antibodies or antigen-binding fragments that can be derivatized (e.g., by being attached to another molecule, e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to PRLR. Thus, the antibodies or antigen-binding fragments thereof of the present invention are intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present invention may be attached to one or more other molecular groups to form bispecific antibodies, detection reagents, pharmaceutical reagents, and / or proteins or polypeptides that can mediate binding of the antibody or antigen-binding fragment to another molecule (e.g., avidin or polyhistidine tags).

[0079] One type of derivatized antibody is a labeled antibody. For example, an antibody or antigen-binding fragment thereof of the present invention may be linked to a detectable marker. A detectable marker according to the present invention may be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such markers are well known in the art and include, for example, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radioisotopes (e.g., 3 H, 125 I, 35 S, 14 C or 32Examples of suitable markers include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., Dynabeads®), heat-generating markers (e.g., gold colloids or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads), and biotin used to bind to avidin (e.g., streptavidin) modified with the marker. Patents teaching the use of such markers include, but are not limited to, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, all of which are incorporated herein by reference. The detectable markers can be detected by methods known in the art. For example, radioactive markers can be detected with photographic film or a scintillation detector, and fluorescent markers can be detected with a photodetector that detects emitted light. Enzymatic markers are generally detected by providing the enzyme with a substrate and detecting the reaction product produced by the enzyme acting on the substrate, and exothermic markers can be detected by simply visualizing the colored marker. In some embodiments, such markers can be used for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable marker may be attached to the antibody or antigen-binding fragment thereof of the present invention via linkers of different lengths to reduce potential steric hindrance.

[0080] The antibodies or antigen-binding fragments thereof of the present invention may also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or glycosyl groups. These groups may be used to improve the biological properties of the antibody, such as increasing serum half-life.

[0081] The present invention provides a multispecific antibody comprising a first antibody or a fragment thereof, another antibody or a fragment thereof, or an antibody mimetic as another antibody derivative, wherein the first antibody or a fragment thereof, another antibody or a fragment thereof, or an antibody mimetic retains its original binding specificity. The first antibody or a fragment thereof is any (monoclonal) antibody or antigen-binding fragment thereof of the present invention that binds to TSLP. As used herein, the term "antibody mimetic" refers to an antibody that specifically binds to an antigen in the same manner as an antibody but does not have an antibody structure. Generally, an antibody mimetic is an artificial peptide or protein with a molar mass of approximately 3 to 20 kDa, such as a designed ankyrin repeat protein (DARPin) or a fynomer. The designed ankyrin repeat protein (DARPin) may be linked to an IgG antibody, an scFv-Fc antibody fragment, or a combination thereof, as described in Patent CN104341529A. As described in patent WO2015141862A1, an anti-IL-17a fynomer is fused to an anti-IL-6R antibody to generate a bispecific fusion polypeptide.

[0082] In some embodiments, the multispecific antibody is formed by conjugating a first antibody or antigen-binding fragment thereof to another antibody or antigen-binding fragment thereof, or antibody mimetic, each of which retains its original binding specificity, and the first antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof described in the present invention. In some embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0083] (antibody drug conjugates) The antibody-drug conjugate comprises an antigen-binding protein portion provided by the present invention and a binding portion. The antigen-binding protein portion includes the above-mentioned "antigen-binding protein" and an antigen-binding fragment obtained from the "antigen-binding protein." Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb. The binding portion may comprise at least one payload. The payload may include an active pharmaceutical ingredient and / or the labeling molecule. The payload is a pharmaceutically active small molecule compound or toxin or other drug molecule form, and may be, but is not limited to, a small molecule compound, a toxin molecule, an oligonucleotide, a proteolysis-targeting chimeric molecule (PROTAC), an affinity ligand, a fluorescent group, a nuclide, etc. Various payloads are known in the art. For example, small molecule compounds generally refer to a series of substances with strong cytotoxicity. Exemplary small molecule compounds can exert their cytotoxic and cell growth inhibitory effects through mechanisms including, but not limited to, tubulin binding, DNA binding, inhibition of RNA polymerase, inhibition of protein synthesis or topoisomerase. For example, the small molecule compound may be a tubulin inhibitor, such as a maytansinoid (e.g., DM1 or DM4), an auristatin (e.g., MMAE or MMAF), or the like. For example, the small molecule compound may be a DNA damaging agent, such as a calicheamicin or a pyrrolobenzodiazepine (PBD). For example, proteolysis-inducing chimeric molecules (PROTACs) are a class of compounds that can cause degradation of target proteins by inducing polyubiquitination of the target protein, and the PROTAC may be a BET protein degrader. The antibody-drug conjugate may further comprise at least one linker. For example, the linker may comprise a cleavable linker or a non-cleavable linker. The linker is used to connect one or more payloads to the antigen-binding protein.In the present application, the cleavable linker may be a "cleavable" linker that facilitates drug release. For example, cleavable linkers include, but are not limited to, acid-sensitive linkers, protease-sensitive linkers, photosensitive linkers, or disulfide-containing linkers. The linker may contain one or more linker components, and various linker components are known in the art, such as maleimidocaproyl (MC), maleimidylpropionyl (MP), valine-citrulline (val-cit, vc, or valine-citrulline), and paminobenzyloxycarbonyl (PAB).

[0084] (Nucleic acids, vectors, host cells and antibody production) The antigen-binding proteins of the present invention may be produced by various methods known in the art, such as recombinant genetic engineering techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector, which is then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibody of the present invention.

[0085] In some specific embodiments, the present invention provides isolated nucleic acid molecules comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present invention, or the heavy chain variable region and / or light chain variable region thereof, or one or more CDRs thereof. In some embodiments, the nucleotide sequence may be one that has been substituted based on codon degeneracy, as known in the art. In some embodiments, the nucleotide sequence is codon-optimized.

[0086] In some embodiments, the present invention provides a cloning vector or expression vector comprising the isolated nucleic acid molecule of the present invention. In some embodiments, the vector is, for example, a plasmid, cosmid, phage, lentivirus, etc. In some embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the present invention in vivo in a subject (e.g., a mammal (e.g., a human)). In some embodiments, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention. The host cell may be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., E. coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell of the present invention is a mammalian cell, such as a CHO cell (e.g., CHO-K1, CHO-S, CHO DXB11, or CHO DG44).

[0087] In some embodiments, the invention provides a method for producing an antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0088] (Pharmaceutical uses, methods of treatment, pharmaceutical compositions and administration devices) The present invention provides uses of the PRLR antigen-binding proteins, antigen-binding protein derivatives, multispecific antibodies, immune cells, and antibody-drug conjugates in the manufacture of PRLR blockers and drugs for preventing and / or treating PRLR-positive diseases.

[0089] Accordingly, the present invention provides methods for blocking PRLR and preventing and / or treating PRLR-positive diseases using the PRLR antigen-binding proteins, antigen-binding protein derivatives, multispecific antibodies, immune cells, and antibody-drug conjugates.

[0090] In some embodiments, the present invention provides pharmaceutical compositions for treatment comprising the PRLR antigen-binding protein, nucleic acid molecule, vector, host cell, immune cell, antigen-binding protein derivative, multispecific antibody and / or antibody-drug conjugate, and optionally a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" includes, but is not limited to, a diluent, excipient, filler, wetting agent, disintegrant, flavoring agent, and binder.

[0091] In some embodiments, the pharmaceutical composition further comprises a co-therapeutic agent, including, but not limited to, a chemotherapeutic agent, a radiotherapeutic agent, an immunosuppressant, or a cytotoxic agent.

[0092] In some embodiments, the antigen-binding protein of the present invention and the combination therapeutic agent are provided as separate components in the pharmaceutical composition or as components of a single composition. Thus, the antigen-binding protein of the present invention and the combination therapeutic agent may be administered in combination or separately, and may be administered simultaneously or sequentially. The antibody against PRLR or its antigen-binding fragment may be administered alone or in combination with other therapeutic agents. The antibody against PRLR or its antigen-binding fragment and one or more other therapeutic agents may be administered separately, simultaneously, or sequentially.

[0093] The pharmaceutical composition may be in any suitable form (determined by the intended mode of administration to the patient), and the PRLR antibody of the present invention may be administered to the patient by a variety of routes (e.g., oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, topical, intramedullary, intracerebroventricular). In any particular situation, the optimal route of administration will be determined by the particular antibody, the individual, the characteristics and severity of the disease, and the individual's health status.

[0094] In some embodiments, the present invention also provides an administration device for administering said antigen binding proteins, antigen binding protein derivatives, multispecific antibodies, immune cells, antibody drug conjugates and pharmaceutical compositions comprising said components, said device comprising: (i) an infusion module used to administer a pharmaceutical composition containing an active ingredient to a subject; (ii) a pharmaceutical composition to be injected, the pharmaceutical composition comprising an active ingredient selected from the group consisting of an antigen-binding protein, an antigen-binding protein derivative, a multispecific antibody, an immune cell, an antibody-drug conjugate, or a combination thereof; (iii) optionally, a medication monitoring module.

[0095] In another preferred embodiment, the administration includes oral administration and parenteral administration. In another preferred embodiment, the parenteral administration includes injection administration, and applicable injection routes include intravenous, intramuscular, intraarterial, intrathecal, intracystic, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal injection, and bolus administration. In another preferred embodiment, the parenteral administration includes topical, epidermal, or transmucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration. In another preferred embodiment, the infusion module is a needleless hypodermic injection device, a microinfusion pump, a transdermal administration device, a bolus administration device, or an osmotic device.

[0096] (Detection method, kit) The antibody or antigen-binding fragment thereof of the present invention can bind to PRLR and can therefore be used to detect the presence or amount of PRLR in a sample.

[0097] In one aspect, the present invention provides a kit comprising an antigen-binding protein of the present invention. In some embodiments, the antigen-binding protein of the present invention comprises a detectable marker. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody of the present invention or an antigen-binding fragment thereof. Preferably, the second antibody further comprises a detectable marker.

[0098] In the present invention, the detectable marker may be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Particularly preferably, such markers can be used for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).

[0099] The present invention provides for the detection of PRLR expression, comprising contacting one or more PRLR antibodies of the present invention (optionally conjugated to a detectable moiety) with a biological sample (individual cells, tissues, or bodily fluids) and detecting whether the sample is positive for PRLR expression or whether the sample has unchanged (e.g., decreased or increased) expression compared to a control sample. In some examples, the tissue or bodily fluid is peripheral blood, leukocytes in peripheral blood, biopsied tissue (e.g., lung or skin biopsy tissue), and tissue. The method can be used for diagnostic or non-diagnostic purposes (e.g., PRLR pathway research, drug screening, immunohistochemical analysis, etc.). In some embodiments, the sample used for non-diagnostic purposes is a cell sample, such as a cell line or ex vivo cell culture.

[0100] In one embodiment, the present invention provides a method for detecting the presence or amount of PRLR in a sample, comprising contacting the sample with an antibody or its antigen-binding fragment described in the present invention under conditions that allow the antibody or its antigen-binding fragment to form a complex with PRLR, and detecting the formation of the complex.

[0101] In another aspect, the present invention provides a method for diagnosing a PRLR-positive disease in a subject, comprising contacting an antibody or antigen-binding fragment thereof, multispecific antibody, or antibody-drug conjugate described in the present invention with a sample derived from the subject under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and PRLR, and detecting the formation of the complex, wherein elevated amounts of PRLR compared to healthy controls indicate a disease such as tumor or alopecia.

[0102] Optionally, the subject is a mammal, including non-human mammals and humans. Preferably, the subject is a human.

[0103] Preferably, the tumor includes, but is not limited to, breast cancer, prostate cancer, ovarian cancer, lung cancer, skin cancer, pancreatic cancer, kidney cancer, stomach cancer, etc.

[0104] In another aspect, the present invention provides a detection kit comprising the PRLR antigen-binding protein, antigen-binding protein derivative, etc. described in the present invention, and in a preferred embodiment, the kit includes instructions for use that describe how to use the kit.

[0105] Example 1: Antigen-immunized animals, preparation of hybridomas, and screening 1.1 Immunization of mice Balb / c mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were immunized by subcutaneous injection with a commercially available PRLR-ECD protein (purchased from Acro, catalog number PRR-H52Ha, amino acid sequence of which is set forth in SEQ ID NO: 1). On day 1, human PRLR-ECD protein was emulsified in complete Freund's adjuvant (Sigma) and then injected subcutaneously into the backs of Balb / c mice (50 μg human PRLR-ECD per mouse, 0.5 mL). On day 14, human PRLR-ECD protein was emulsified in incomplete Freund's adjuvant (Sigma) and then injected subcutaneously into the backs of Balb / c mice (30 μg human PRLR-ECD per mouse, 0.5 mL). On day 28, human PRLR-ECD protein was emulsified in incomplete Freund's adjuvant and then injected subcutaneously into the backs of Balb / c mice (30 μg human PRLR-ECD per mouse, 0.5 mL). Three weeks later, 30 μg of PRLR-ECD was injected into the tail vein for a boost. Three to four days later, the mice's spleens were removed and fusion experiments were performed.

[0106] 1.2 Preparation and screening of hybridoma cells Three to four days after the final booster immunization, mouse splenocytes were fused with mouse myeloma cells SP2 / 0 by PEG using standard hybridoma technology. The fused cells were homogeneously mixed in complete medium (RPMI 1640 medium, GlutaMax, 1% penicillin-streptomycin, 1x HAT, 20% FBS (all products purchased from Gibco)) and 3x10 6 The cells were cultured in 30 96-well culture plates at 200 μL / well. After 7 to 12 days, the supernatant was collected and hybridoma wells positive for human PRLR-ECD binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA). Hybridoma wells positive for human PRLR-ECD binding were subjected to first and second subcloning by limiting dilution to obtain the desired hybridoma cell line, designated SPGA02-260.

[0107] Here, hybridoma wells positive for human PRLR-ECD binding activity were screened by indirect enzyme-linked immunosorbent assay as follows: Recombinant human PRLR-ECD protein was diluted to 0.5 μg / mL in coating solution (phosphate buffer, pH 7.4) and added to an ELISA plate at 100 μL / well for overnight coating at 4°C. The plate was washed once with PBST, and blocking solution (1% BSA-PBST) was added at 300 μL / well. The plate was then incubated at 37°C for 2 hours. Afterwards, the plate was washed three times with PBST for use. The collected hybridoma supernatants were sequentially added to the blocked ELISA plate at 100 μL / well and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-conjugated goat anti-mouse IgG secondary antibody (purchased from Abcam, catalog number ab6789) was added and incubated at 37°C for 30 minutes. The plate was then washed three times with PBST and tapped against absorbent paper to remove as much residual liquid as possible. 100 μL of TMB (purchased from KPL, catalog number 52-00-03) was added to each well and the plate was incubated at room temperature (20±5°C) in the dark for 5 minutes. 50 μL of 2M H2SO4 stop solution was added to each well to stop the substrate reaction. The OD value at 450 nm was read using a microplate reader to analyze the binding ability of the test hybridoma supernatants to the target antigen PRLR-ECD.

[0108] Example 2: Large-scale preparation and identification of mouse anti-human PRLR monoclonal antibodies The screened hybridoma cell lines were expanded in serum-containing complete medium, centrifuged, and replaced with serum-free medium (SFM) to a cell density of 1–2 × 10 7 The culture supernatant was obtained by centrifugation and purified by Protein A affinity chromatography to obtain the mouse anti-human PRLR-ECD protein monoclonal antibody SPGA02-260.

[0109] Example 3: Binding activity of mouse monoclonal antibodies against human PRLR-ECD The binding ability of mouse antibodies to human PRLR was measured by indirect enzyme-linked immunosorbent assay (ELISA). Recombinant human PRLR-ECD protein was diluted to 0.5 μg / mL in coating solution (phosphate buffer, pH 7.4) and added to ELISA plates at 100 μL / well for overnight coating at 4°C. The plates were washed once with PBST, and 300 μL / well of blocking solution (1% BSA-PBST) was added. The plates were then incubated at 37°C for 2 hours, after which they were washed three times with PBST. Purified antibodies were diluted to 1 μg / mL in 1% BSA-PBST. After a 1:3 gradient dilution, 100 μL / well of the antibodies were added to ELISA plates and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-conjugated goat anti-mouse IgG secondary antibody (purchased from Abcam, catalog number ab6789) was added and incubated at 37°C for 30 minutes. The plate was then washed three times with PBST and tapped against absorbent paper to remove as much residual liquid as possible. 100 μL of TMB (purchased from KPL, catalog number 52-00-03) was added to each well and the plate was incubated at room temperature (20±5°C) in the dark for 5 minutes. 50 μL of 2M H2SO4 stop solution was added to each well to stop the substrate reaction. The OD value at 450 nm was read using a microplate reader to analyze the binding ability of the test antibody to the target antigen PRLR-His.

[0110] The results are shown in Figure 1. The results revealed that SPGA02-260 bound to human PRLR-ECD with high affinity, and EC 50 was 20.9 ng / mL and 0.14 nM.

[0111] Example 4: Measurement of binding affinity of mouse antibodies to human breast cancer cells T47D In this example, the binding affinity of SPGA02-260 to human breast cancer cells T47D was measured by fluorescence activated cell sorting (FACS).

[0112] In this experiment, human breast cancer cell line T47D was used as the target cell, and SPGA02-260 was used as the primary antibody. SPGA02-260 was serially diluted six times starting from 20 μg / mL. 100 μL of each diluted antibody was incubated with 100 μL of T47D cells at room temperature for 30 minutes. (The cells were suspended in 100 μL of RPMI-1640 serum-free medium (purchased from Gibco, catalog number 61870036). The maximum working concentration of the monoclonal antibody was 10 μg / mL.) The cells were washed twice with PBS to remove unbound SPGA02-260. Then, the cells were incubated with 50 μL of 5 μg / mL Alexa Fluor ... The cells were incubated with 488-labeled anti-mouse fluorescent secondary antibody (Thermo, purchased from Invitrogen, catalog number A11001) for 40 minutes at 4°C in the dark, washed twice with PBS to remove unbound secondary antibody, and finally resuspended in 100 μL of PBS. The binding affinity of SPGA02-260 to the cells was measured using a flow cytometer (purchased from Beckman, model CytoFLEX). The obtained data were fitted and analyzed using the software GraphPad Prism 6.

[0113] The results are shown in Figure 2. The results revealed that SPGA02-260 could specifically bind to the human breast cancer cell line T47D, which highly expresses PRLR on the cell surface, and its EC 50 was 26.0 ng / mL and 0.17 nM.

[0114] Example 5: Inhibition of mouse antibodies against PRL-induced signaling In this example, Western blot analysis was used to measure the effect of antibody SPGA02-260 on the downstream transcriptional activator family STAT5 and ERK1 / 2 signaling pathways in human breast cancer cells T47D after stimulation with PRL.

[0115] Logarithmically growing breast cancer cells T47D were digested with trypsin and resuspended in complete medium, and 2 × 10 6Cells were seeded into a 6-well plate at 1 mL per well and incubated overnight at 37°C in a CO2 incubator. The next day, after the cells had adhered to the plate, the medium was removed from the 6-well plate. 2 mL of room-temperature sterile PBS was gently added to each well to wash off any remaining medium, and the PBS was then removed. SPGA02-260 was diluted to concentrations of 20 μg / mL, 2 μg / mL, and 0.2 μg / mL in 1640 medium without fetal bovine serum. 1 mL of each antibody was slowly added to each well and incubated for 30 minutes in the incubator. After 30 minutes, 500 μL of a 500 ng / mL PRL (ACRO, Cat. #PRN-H5257) solution (prepared in 1640 medium) was slowly added to each well. The 6-well plate was gently shaken and placed in a CO2 incubator for further incubation. After 30 minutes of incubation, the 6-well plate was placed on ice, the medium was removed, and each well was gently washed once with 2 mL of pre-chilled sterile PBS. The PBS was removed, and 200 μL of RIPA lysis buffer (TEKNOVA, Cat. #R3792) was added to each well. After uniform mixing, the plate was left to lyse for 10 minutes. The cell lysates in the plate were collected in a centrifuge tube and centrifuged at 13,000 rpm for 10 minutes. The supernatant was quantified by BCA and then stored in a -80°C refrigerator for further use. Phosphorylated STST5 (pSTAT5) and phosphorylated ERK1 / 2 (pERK1 / 2) were measured by standard Western blot analysis.

[0116] pSTAT5 was measured as follows: The cell lysates were subjected to SDS-PAGE electrophoresis (100 μg / lane), transferred to a PVDF membrane by electrophoretic transfer, blocked with 1% BSA-TBST (1 hour at room temperature with shaking), and then incubated with 1 μg / mL (1% BSA-TBST) rabbit anti-human phosphorylated STAT5 (Tyr694) antibody (purchased from CST, Cat# 94205S) at 1 μg / mL. The mixture was incubated with shaking at room temperature for 2 hours. After washing three times with TBST, HRP-conjugated goat anti-rabbit IgG secondary antibody (purchased from Abcam, Cat# ab97051, diluted 1:10,000 with 1% BSA-TBST according to the manufacturer's instructions) was added. The mixture was incubated with shaking at room temperature for 1 hour. After washing three times with TBST, the PVDF membrane was coated with an appropriate amount of Pierce™ ECL Western Blotting gel. Substrate solution (purchased from Thermo, catalog number 32209) was added dropwise, and automatic imaging was performed at room temperature in the dark using a biomolecular imager (purchased from Thermo, model CL1500).

[0117] pERK1 / 2 was measured as follows: The cell lysate was subjected to SDS-PAGE electrophoresis (100 μg / lane), then electrophoretically transferred to a PVDF membrane. The membrane was blocked with 1% BSA-TBST (1 hour at room temperature with shaking), and then 1 μg / mL (1% BSA-TBST diluted) rabbit anti-phospho-p44 / 42 MAPK (Erk1 / 2, Thr202 / Tyr204) antibody (Cell Signaling Technology, Cat. #9101) was added. The membrane was incubated with shaking at room temperature for 2 hours. After washing three times with TBST, an HRP-conjugated goat anti-rabbit IgG secondary antibody (Abcam, Cat. #ab97051, diluted 1:10,000 with 1% BSA-TBST according to the manufacturer's instructions) was added. The membrane was incubated with shaking at room temperature for 1 hour. After washing three times with TBST, the PVDF membrane was coated with an appropriate amount of Pierce™ ECL Western Blotting gel. Substrate solution (purchased from Thermo, catalog number 32209) was added dropwise, and automatic imaging was performed at room temperature in the dark using a biomolecular imager (purchased from Thermo, model CL1500).

[0118] β-actin was measured as follows: The cell lysate was subjected to SDS-PAGE electrophoresis (100 μg / lane), then electrophoretically transferred to a PVDF membrane. The membrane was blocked with 1% BSA-TBST (1 hour at room temperature with shaking). A 1 μg / mL (1% BSA-TBST diluted) mouse anti-human β-actin monoclonal antibody (Thermo, Cat. #MA1-140) was added and incubated with shaking at room temperature for 2 hours. After washing three times with TBST, an HRP-conjugated goat anti-mouse IgG secondary antibody (Beijing Boaolun Immunotechnology Co., Ltd., Cat. #BF03001, diluted 1:10,000 with 1% BSA-TBST according to the manufacturer's instructions) was added and incubated with shaking at room temperature for 1 hour. After washing three times with TBST, the PVDF membrane was coated with an appropriate amount of Pierce™ ECL Western Blotting gel. Substrate solution (purchased from Thermo, catalog number 32209) was added dropwise, and automatic imaging was performed at room temperature in the dark using a biomolecular imager (purchased from Thermo, model CL1500).

[0119] The results are shown in Figure 3. The β-actin content in each measured sample was consistent as a control. During the experiment, STAT5 was not phosphorylated in T47D cells without PRL stimulation, but was clearly phosphorylated with PRL. The SPGA02-260 antibody completely inhibited the PRL-stimulated STAT5 phosphorylation at concentrations of 20 μg / mL and 2 μg / mL, and still partially inhibited STAT5 phosphorylation at a concentration of 0.2 μg / mL.

[0120] The results are shown in Figure 4. The content of β-actin was consistent in each measured sample as a control. During the experiment, ERK1 / 2 was not phosphorylated in T47D cells without PRL stimulation, but was clearly phosphorylated with PRL. SPGA02-260 was able to completely inhibit the PRL-stimulated ERK1 / 2 phosphorylation at concentrations of 20 μg / mL, 2 μg / mL, and 0.2 μg / mL.

[0121] Example 6: Endocytosis of mouse antibodies by target cells T47D In this example, SPGA02-260-induced endocytosis in human breast cancer cells T47D was measured by fluorescence activated cell sorting (FACS).

[0122] The experimental method was as follows: 3 × 10 T47D cells were cultured. 5 The cells were seeded at 1000 cells / well in a 24-well culture plate. The next day, the supernatant was discarded, the cells were washed once with PBS, and 0.5 mL of culture medium containing 10 μg / mL SPGA02-260 was added to each well. The cells were incubated at 37°C for 0, 1, 3, and 6 hours, respectively. The supernatant was discarded, the cells were digested with trypsin, transferred to a centrifuge tube, washed once with PBS, and added with 5 μg / mL Alexa Fluor 488-labeled anti-mouse fluorescent secondary antibody (Thermo, purchased from Invitrogen, catalog number A11001). The cells were incubated at 4°C in the dark for 40 minutes, washed once with PBS, and resuspended in 100 μL of PBS for flow cytometry.

[0123] The results are shown in Figure 5. The results revealed that after antibody SPGA02-260 bound to the PRLR antigen on the surface of T47D cells, endocytosis occurred in a time-dependent manner, and after 3 hours, approximately 50% of PRLR had been endocytosed.

[0124] Example 7: Production of chimeric antibodies In this example, the heavy chain variable region and light chain variable region of SPGA02-260 were obtained by molecular biological techniques, and further, a chimeric antibody SPGA02-ch260 was constructed.

[0125] JPEG0007827890000002.jpg40124

[0126] Specific information on related sequences is provided in Table 1 above, except that The SPGA02-260 heavy chain variable region gene sequence was 360 bp in length, encoded 120 amino acid residues, and had the amino acid sequence set forth in SEQ ID NO: 2 and the nucleotide sequence set forth in SEQ ID NO: 3. The light chain variable region gene sequence was 321 bp in length, encoded 107 amino acid residues, and had the amino acid sequence set forth in SEQ ID NO: 4 and the nucleotide sequence set forth in SEQ ID NO: 5.

[0127] The obtained heavy chain variable region amino acid sequence was spliced ​​with the human IgG1 constant region (amino acid sequence set forth in SEQ ID NO: 6), and the light chain variable region was spliced ​​with the human kappa chain constant region (amino acid sequence set forth in SEQ ID NO: 7) to construct the heavy chain (amino acid sequence set forth in SEQ ID NO: 8) and light chain (amino acid sequence set forth in SEQ ID NO: 9) of SPGA02-ch260. Each was constructed into the pcDNA3.4 expression vector and transfected into Expi293F cells. The resulting chimeric antibody SPGA02-ch260 was purified, and SDS-PAGE electrophoresis revealed that the expressed antibody had the correct molecular weight and an antibody purity of over 95%. After quantification, the antibody was stored at 4°C for use.

[0128] Example 8: Determining the affinity of chimeric antibodies for target antigens In this example, the affinity of the chimeric antibody SPGA02-ch260 for human PRLR-ECD was measured by ELISA.

[0129] The experimental method was as described in Example 3.

[0130] The results are shown in Figure 6. SPGA02-ch260 was found to bind to PRLR with high affinity, and EC 50 was 12.9 ng / mL and 0.09 nM.

[0131] Example 9: Determination of binding affinity of chimeric antibodies to target cells In this example, the binding affinity of the chimeric antibody SPGA01-ch260 to human breast cancer cells T47D was measured by the FACS method.

[0132] The experimental method was as described in Example 4.

[0133] The results are shown in Figure 7. SPGA02-ch260 was found to bind with high affinity to T47D cells, which highly express PRLR, and EC 50 was 31.0 ng / mL and 0.21 nM.

[0134] Example 10: Measurement of binding of chimeric antibodies to mouse PRLR In this example, the difference in binding of SPGA02-ch260 to human PRLR and mouse PRLR was measured by ELISA.

[0135] Mouse PRLR protein (purchased from Sino Biological Inc., catalog number 50457-M08H), i.e., mouse PRLR, was used to coat a 96-well ELISA plate at 0.5 μg / well to measure the cross-reactivity of SPGA02-ch18 and SPGA02-ch260 with mouse PRLR. The rest of the specific experimental procedures were as described in Example 8.

[0136] The results are shown in Figure 8. SPGA02-ch260 was found to be able to bind to mouse PRLR protein, and EC 50 The binding capacity was 297.3 ng / mL, which was clearly lower than that of human PRLR (EC 50 is 12.9ng / mL).

[0137] Example 11: Determination of the target antigen-binding region of antibody SPGA02-260 In this example, the epitope of SPGA02-260 that binds to PRLR was measured by Western blot and ELISA.

[0138] To determine the epitope of SPHA02-260 that binds to human PRLR, we searched the literature and NCBI to obtain the human PRLR extracellular domain (PRLR-ECD) gene (amino acids 1 to 234 of NCBI accession number NP_000940.1; the amino acid sequence is set forth in SEQ ID NO: 1, except that the amino acid sequence of the functional domain, fibronectin type-III 1 (amino acids 27 to 128), is set forth in SEQ ID NO: 10, and the amino acid sequence of the functional domain, fibronectin type-III 2 (amino acids 129 to 229), is set forth in SEQ ID NO: 11). Fc fusion proteins of the two functional domains of PRLR-ECD, namely, fibronectin type-III 1 and fibronectin type-III 2, were expressed.

[0139] Reduced and denatured human PRLR and recombinantly expressed two functional domains, namely, fibronectin type III 1 and fibronectin type III 2, were subjected to SDS-PAGE electrophoresis (400 ng / lane), and then transferred to a PVDF membrane by electrophoretic transfer. The membrane was blocked with 1% BSA-TBST (1 hour at room temperature with shaking), and then 1 μg / mL of mouse antibody SPGA02-260 (diluted in 1% BSA-TBST) was added, and the membrane was incubated with shaking at room temperature for 2 hours. After washing three times with TBST, an HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Abcam, catalog number ab6789, diluted 1:10,000 with 1% BSA-TBST according to the manufacturer's instructions) was added, and the membrane was incubated with shaking at room temperature for 1 hour. After washing three times with TBST, the membrane was coated with an appropriate amount of Pierce™ ECL Western Blotting gel. Substrate solution (purchased from Thermo, catalog number 32209) was added dropwise, and automatic imaging was performed at room temperature in the dark using a biomolecular imager (purchased from Thermo, model CL1500).

[0140] The results are shown in Figure 9. SPGA02-260 was able to specifically bind to reduced and denatured human PRLR, and SPGA02-260 was detected to bind to PRLR via the second functional domain of human PRLR-ECD, i.e., fibronectin type III 2.

[0141] Human PRLR-ECD-fibronectin type-III 2 was digested to obtain 10 partially overlapping polypeptides, each with the following amino acid sequence: 1:bio-PPLELAVEVKQPEDRKPYLW(129~148) 2:bio-QPEDRKPYLWIKWSPPTLID(139~158) 3:bio-IKWSPPTLIDLKTGWFTLLY(149~168) 4:bio-LKTGWFTLLYEIRLKPEKAA(159~178) 5:bio-EIRLKPEKAAEWEIHFAGQQ(169~188) 6:bio-EWEIHFAGQQTEFKILSLHP(179~198) 7:bio-TEFKILSLHPGQKYLVQVRC(189~208) 8:bio-GQKYLVQVRCKPDHGYWSAW(199~218) 9:bio-KPDHGYWSAWSPATFIQIPS(209~228) 10:bio-SPATFIQIPSDFTMND(219~234)

[0142] The binding status of SPGA02-260 to the above 10 polypeptides was measured using ELISA to determine the epitope of SPGA02-260 that binds to PRLR.

[0143] The results are shown in Figure 10. SPGA02-260 bound only to the above-mentioned polypeptide 7, i.e., the polypeptide consisting of amino acids 189 to 208 at the N-terminus of the human PRLR protein, indicating that the binding epitope of SPGA02-260 is located between G189 and C208 of the human PRLR protein.

[0144] Example 12: Humanization of antibody SPGA02-260 The amino acid sequences of the light and heavy chain variable regions of the candidate mouse antibody SPGA02-260 were analyzed, and three antigen complementarity-determining regions (CDRs) and four framework regions (FRs) of the mouse antibody were determined based on the Kabat rules. The amino acid sequences of the SPGA02-260 heavy chain complementarity-determining regions were HCDR1: TVSGFSLTRNGV (SEQ ID NO: 12), HCDR2: IWGDGST (SEQ ID NO: 13), and HCDR3: AKEGLYYYGRYFDV (SEQ ID NO: 14). The amino acid sequences of the light chain complementarity-determining regions were LCDR1: KASQDVGSAV (SEQ ID NO: 15), LCDR2: WASTRHT (SEQ ID NO: 16), and LCDR3: QQYSNYPLT (SEQ ID NO: 17).

[0145] The humanized template that best matched each of the mouse antibody FR regions was selected from the Germline database. The CDR regions of the mouse antibody were then grafted onto the selected humanized template to replace the CDR regions of the human template to form a humanized antibody heavy chain variable region (SPGA02-hu260VH, SEQ ID NO: 18). This was then recombined with a human IgG1 constant region (containing the S228P mutation, SEQ ID NO: 6) to form a humanized monoclonal antibody heavy chain (SPGA02-hu260H, SEQ ID NO: 20). The humanized antibody light chain variable region (SPGA02-hu260VL, SEQ ID NO: 19) was recombined with a human kappa chain constant region to form a humanized light chain (SPGA02-hu260L, SEQ ID NO: 21). The heavy and light chains of the humanized antibodies were constructed in the pcDNA3.4 expression vector, transfected into Expi-293F cells, and purified with Protein A to obtain the humanized antibody SPGA02-hu260. SDS-PAGE electrophoresis and SEC-HPLC revealed that each SPGA02-260 antibody had the correct molecular weight and a purity of over 95%.

[0146] Example 13: Determining the affinity of humanized antibody SPGA02-hu260 for its target antigen In this example, the affinity of the humanized antibody SPGA02-hu260 for human PRLR-ECD was measured by ELISA.

[0147] The experimental method was as described in Example 3.

[0148] The results are shown in Figure 11. SPGA02-hu260 was found to bind to PRLR with high affinity, and EC 50 was 8.98 ng / mL, or 0.06 nM.

[0149] Example 14: Inhibition of signal transduction induced by binding of PRLR to PRL by humanized antibody SPGA02-hu260 The experimental method was the same as in Example 5. The working concentrations of antibody SPGA02-hu260 were adjusted to 1.0 / 0.5 / 0.25 / 0.1 μg / mL.

[0150] The results are shown in Figures 12 and 13. In each measured sample, the content of β-actin was matched as a control.

[0151] In T47D cells, STAT5 was clearly phosphorylated by the action of PRL. The antibody SPGA02-hu260 completely inhibited the PRL-induced STAT5 phosphorylation in T47D cells at concentrations of 1 μg / mL and 0.5 μg / mL, but partially inhibited the PRL-induced STAT5 phosphorylation at a concentration of 0.25 μg / mL.

[0152] In T47D, ERK1 / 2 was clearly phosphorylated by the action of PRL, and the SPGA02-hu260 antibody could still completely inhibit the PRL-stimulated ERK1 / 2 phosphorylation even at a concentration of 0.1 μg / mL.

[0153] Example 15: Humanized antibody SPGA02-hu260 inhibits signal transduction induced by PRLR-GH binding The experimental method was almost the same as in Example 5. Instead of human PRL, 1 μg / mL human growth hormone (GH1, SinoBiological, Cat#16122-HNCE) was used as a stimulator to induce PRLR signaling.

[0154] The results are shown in Figures 14 and 15. In each measured sample, the content of β-actin was matched as a control.

[0155] In T47D cells, STAT5 was clearly phosphorylated by GH1 stimulation. The antibody significantly inhibited GH1-induced STAT5 phosphorylation in T47D cells at concentrations of 20 μg / mL and 2 μg / mL, but this inhibitory effect was almost abolished at a concentration of 0.2 μg / mL.

[0156] In T47D, GH1-induced ERK1 / 2 phosphorylation was clearly observed, and the SPGA02-hu260 antibody could still significantly inhibit the GH1-induced ERK1 / 2 phosphorylation even at a concentration of 0.2 μg / mL.

[0157] Example 16: Inhibition of proliferation of BAF3-huPRLR cells by antibody SPGA02-hu260 Logarithmically growing mouse B cells, BAF3, were cultured in 1640 culture medium (complete medium) containing 10 ng / mL mouse IL3 (Beyotime Biotech Inc., P5912) and 10% FBS (Gibico). The cells were infected with a lentivirus overexpressing human PRLR (Jiman Biotechnology (Shanghai) Co., Ltd., catalog number GM-10162OL01). Screening and subcloning were performed in the presence of 5 μg / mL puromycin dihydrochloride (Jiman Biotechnology (Shanghai) Co., Ltd., catalog number GM-040401-1) to obtain a cell line stably overexpressing human PRLR, i.e., BAF3-huPRLR.

[0158] BAF3-huPRLR cells were cultured in 1640 cell culture medium containing 40 ng / mL human PRL (ACRO Biosystems Co., Ltd., PRR-H52Ha) at a concentration of 2 × 10 5The antibody SPGA02-hu260 was diluted to 30 μg / mL in 1640 medium without PRL, and then diluted 10-fold in eight gradients. Each dilution was added to the 96-well cell culture plate containing the BAF3-huPRLR cells at 100 μL / well. The cells were then cultured in a CO2 incubator for 72 hours, after which CCK8 reagent was added according to the manufacturer's instructions. After incubation at 37°C for 2 hours, the OD 450 was measured.

[0159] The results are shown in Figure 16. SPGA02-hu260 could effectively inhibit the proliferation of BAF3-huPRLR cells stimulated by human PRL, and the IC 50 was 8.28 ng / mL, or 0.06 nM.

[0160] The above-mentioned examples merely illustrate some embodiments of the present invention, and although they are specifically and in detail described, they should not be construed as limiting the scope of the present invention. Those skilled in the art may make modifications and improvements without departing from the concept of the present invention, and all of these modifications and improvements fall within the scope of the present invention. Therefore, the scope of the present invention is governed by the claims set forth below.

Claims

1. The following complementarity determining regions: (1) Heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence represented by SEQ ID NO: 12 (2) Heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence represented by SEQ ID NO: 13 (3) Heavy chain complementarity-determining region 3 (HCDR3) containing the amino acid sequence represented by SEQ ID NO: 14 (4) Light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence represented by SEQ ID NO: 15 (5) Light chain complementarity-determining region 2 (LCDR2) comprising the amino acid sequence represented by SEQ ID NO: 16 (6) A PRLR antigen-binding protein comprising a light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO:

17.

2. the antigen-binding protein 2. The antigen-binding protein of claim 1, comprising a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 2 and a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO:

4.

3. 3. The antigen-binding protein of claim 2, further comprising a human IgG1 constant region and a human kappa (kappa) chain constant region, wherein the amino acid sequence of the human IgG1 constant region is as set forth in SEQ ID NO: 6, the amino acid sequence of the human kappa (kappa) chain constant region is as set forth in SEQ ID NO: 7, the VH of the PRLR antigen-binding protein is linked to the human IgG1 constant region to form a heavy chain, and the VL of the PRLR antigen-binding protein is linked to the human kappa (kappa) chain constant region to form a light chain.

4. the antigen-binding protein 4. The antigen-binding protein of claim 3, comprising a heavy chain HC comprising the amino acid sequence shown in SEQ ID NO:8 and a light chain LC comprising the amino acid sequence shown in SEQ ID NO:

9.

5. A humanized PRLR antigen-binding protein, characterized in that it comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 18 and a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO:

19.

6. the humanized PRLR antigen binding protein 6. The humanized PRLR antigen binding protein of claim 5, comprising a heavy chain HC comprising the amino acid sequence shown in SEQ ID NO: 20 and a light chain LC comprising the amino acid sequence shown in SEQ ID NO:

21.

7. A nucleic acid molecule encoding the PRLR antigen-binding protein of claim 1 or the humanized PRLR antigen-binding protein of claim 5.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell containing the nucleic acid molecule described in claim 7.

10. A pharmaceutical composition comprising the PRLR antigen-binding protein of claim 1 and / or the humanized PRLR antigen-binding protein of claim 5.

11. The pharmaceutical composition according to claim 10, which is used for preventing and / or treating a PRLR-positive disease, wherein the PRLR-positive disease is a tumor or alopecia.

12. A method for producing a drug, kit and / or administration device for preventing and / or treating a PRLR-positive disease, using a PRLR antigen-binding protein according to any one of claims 1 to 4.

13. The method according to claim 12, wherein the PRLR-positive disease is a tumor or alopecia.

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