PRLR antigen-binding protein, method for producing the same, and use thereof

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 a PRLR antigen-binding protein, a method for producing the same, and uses thereof.

Background Art

[0002] Prolactin (PRL) is also called lactogenic hormone or mammotropic hormone, and is an anterior pituitary peptide hormone necessary for reproductive success. Prolactin receptor (PRLR), growth hormone receptor (GHR), and placental lactogen receptor (PLR) constitute a gene family. 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. PRLR is associated with various physiological functions such as cell proliferation, differentiation, lactation, and reproduction.

[0003] Mammalian PRL is known to have at least two forms of receptors, a long form and a short form. The diversity of the PRLR structure is mainly brought about by mutations in the intracellular domain. PRLR is a transmembrane protein with only one transmembrane domain and consists of three parts: an extracellular domain, a transmembrane domain, and an intracellular domain, and has the structural characteristics of the cytokine receptor superfamily. PRLR 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 region, while birds have two repeating units of ligand-binding regions. 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 types of exon 1, exon E13 which is the human counterpart of rat and mouse E13, and a new human exon called E1N. The 5'-untranslated region further contains one universal non-coding exon 2 and part of exon 3, and the latter contains the translation initiation codon. Exons E13 and ElN are less than 800 base pairs. These two exons are expressed in human mammary tissue, breast cancer cells, gonads, and liver. In short, transcripts containing E13 are expressed in most tissues. The PRLR gene product is encoded by exons 3-10, and exon 10 encodes most of the intracellular region. Exons E13 and ElN are each transcribed from either of the two promoters, PIII and PN. The PIII promoter contains Spl and C / EBP elements, which are identical to the elements in the rodent promoter and have 81% homology with the -480 / -106 region of the corresponding gene in rats and mice. The PN promoter contains a putative binding site for ETS family proteins and a half-site for nuclear receptors.

[0004] The activation of PRL-R by the binding of PRL to its receptor to form a trimer is a prerequisite and necessary condition for various biological effects to occur. After activation, PRL-R phosphorylates JAK2, induces the phosphorylation of distal tyrosine within the receptor cell, activates STAT proteins, and the activated STAT translocates into the nucleus to activate the transcription of target genes, thereby obtaining biological effects. This is the basic pattern by which PRL, a mediator of the neuro-endocrine-immune network, regulates immune function. Additionally, after binding to its receptor, PRL can activate the PI3K and MAPK signaling pathways, increase the expression of cyclin D1, and promote the proliferation of breast cancer cells.

[0005] PRLR is highly expressed in various endocrine-related tumor cells such as breast cancer, prostate cancer, and ovarian cancer. It is associated with the occurrence and progression of these tumors. An increase in the circulating concentration of its ligand, prolactin (PRL), is a high-risk factor for the occurrence of breast cancer, and it is also one of the negative correlation indicators when judging the prognosis in breast cancer patients. Recent research has revealed that the activation of PRLR is related to alopecia, and an increase in the circulating prolactin concentration is closely related to alopecia. Therefore, PRLR has become a drug discovery target, and by effectively inhibiting the activation of PRLR, various related diseases can be treated. 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 its method of use. 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 pathological conditions.

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

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention provides a novel PRLR antigen-binding protein, a method for producing the same, and its use to address the above problems. The present invention has obtained a series of novel PRLR antigen-binding proteins with biological functions using hybridoma technology. The PRLR antigen-binding protein has good affinity and binding activity for human PRLR-ECD and inhibits the activation of PRLR. The PRLR antigen-binding protein described in the present invention can be used for treating various PRLR-related diseases such as tumors and alopecia, and provides a new idea for the treatment of PRLR-related diseases.

Means for Solving the Problems

[0008] The technical solution of the present invention for achieving the above invention objective is as follows. In one aspect, the present invention provides a PRLR antigen-binding protein.

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

[0010] Furthermore, specifically, the PRLR antigen-binding protein (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 compared 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 compared 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 substitution is a conservative substitution.

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

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

[0013] Furthermore, specifically, the PRLR antigen-binding protein (1) a heavy chain HC containing the amino acid sequence shown in SEQ ID NO: 8 and / or a light chain LC containing the amino acid sequence shown in SEQ ID NO: 9 or (2) a heavy chain and a light chain, wherein compared with the heavy chain and light chain of (1), the heavy chain has 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 chain has 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, and includes heavy and light chains.

[0014] Furthermore, specifically, the above-mentioned PRLR antigen-binding protein includes a chimeric antibody, a humanized antibody or a fully human antibody.

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

[0016] In another aspect, the present invention provides a humanized PRLR antigen-binding protein, and the humanized PRLR antigen-binding protein (1) A heavy chain variable region VH containing the amino acid sequence shown in SEQ ID NO: 18, and / or a light chain variable region VL containing the amino acid sequence shown in SEQ ID NO: 19 Or (2) 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 compared to the VH of (1), and / or 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 compared to the VL of (1) Or (3) VH having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the VH of (1), and / or VL having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the VL of (1), wherein the substitution is a conservative substitution.

[0017] Specifically, the humanized PRLR antigen-binding protein is (1) A heavy chain HC containing the amino acid sequence shown in SEQ ID NO: 20, and / or a light chain LC containing the amino acid sequence shown in SEQ ID NO: 21 Or (2) a heavy chain and a light chain, wherein the heavy chain has 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 chain and light chain of (1), and / or the light chain has 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 chain and light chain of (1).

[0018] In another aspect, the present invention provides a series of nucleic acid molecules encoding the PRLR antigen-binding protein or the 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 containing one or more of the nucleic acid molecules described in the present application.

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

[0022] In another aspect, the present invention provides a series of host cells containing the above nucleic acid molecules or the above vectors.

[0023] Specifically, the host cells include, but are not limited to, microorganisms, plants, 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, lipofectamin transfection, etc.

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

[0025] In another aspect, the present invention provides immune cells containing the above chimeric antigen receptor.

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

[0027] Specifically, the detectable labeling molecule is an enzyme (e.g., 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-described PRLR antigen-binding protein or humanized PRLR antigen-binding protein and another antibody or a fragment thereof or an antibody mimetic.

[0029] Specifically, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetravalent 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 above-described PRLR antigen-binding protein or humanized PRLR antigen-binding protein, and the binding portion comprises a detectable marker, a drug, a toxin, a cytokine, a radioisotope, an enzyme, or a combination thereof, but is not limited thereto, 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 a pharmaceutical composition comprising the above-described 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] Specifically, the pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier.

[0033] Furthermore, 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 combination therapeutic agent, and the combination therapeutic agent includes, but is not limited to, a chemotherapeutic agent, a radiation therapy 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, the method including culturing the above-mentioned host cell when the antigen-binding protein is expressed.

[0036] In another aspect, the present invention provides the use of 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, antibody-drug conjugate and / or pharmaceutical composition in the manufacture of a PRLR blocker, kit and / or medical device.

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

[0038] In another aspect, the present invention provides the use of 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, antibody-drug conjugate and / or pharmaceutical composition in the manufacture of a drug, kit and / or administration device for preventing and / or treating PRLR-positive diseases.

[0039] Specifically, the above-mentioned PRLR-positive diseases include, but are not limited to, tumors or alopecia.

[0040] Furthermore, specifically, the above-mentioned tumors include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, lung cancer, skin cancer, pancreatic cancer, kidney cancer, gastric cancer, etc.

[0041] In another aspect, the present invention provides the use of the above-mentioned 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 method for detecting PRLR qualitatively or quantitatively using the above-mentioned PRLR antigen-binding protein or humanized PRLR antigen-binding protein, and the detection method is used for purposes other than the diagnosis or treatment of diseases.

[0043] In another aspect, the present invention provides a method for treating PRLR-positive related diseases by administering an effective amount of the above-mentioned PRLR antigen-binding protein, humanized PRLR antigen-binding protein, immune cells, antigen-binding protein derivatives, multispecific antibodies, antibody-drug conjugates and / or pharmaceutical compositions to a subject in need thereof.

[0044] Specifically, the PRLR-positive diseases include tumors or alopecia, etc.

[0045] Furthermore, specifically, the tumors include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, lung cancer, skin cancer, pancreatic cancer, kidney cancer, gastric cancer, etc.

[0046] In another aspect, the present invention provides a kit 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, antibody-drug conjugate and / or pharmaceutical composition, and optionally, an instruction manual.

[0047] In another aspect, the present invention provides an administration device comprising (1) an infusion module used for administering the above-mentioned pharmaceutical composition to a subject in need thereof, and (2) optionally, a drug efficacy monitoring module.

Advantages 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 by hybridoma technology, has good affinity and binding activity for human PRLR-ECD, and can effectively inhibit the activation of PRLR.

[0049] (2) The PRLR antigen-binding protein described in the present invention can be used for treating various PRLR-positive diseases such as tumors and alopecia, and provides new ideas for the treatment of PRLR-positive diseases.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0051] Hereinafter, the present invention will be described in more detail using specific examples. The following examples are only for explaining the present invention and are not for limiting the present invention. The experimental methods used in the following examples are carried out under general normal conditions for experimental methods where specific conditions are not specified in the examples unless otherwise stated. Regarding the materials, reagents, etc. used in the following examples, all can be obtained as commercially available products unless otherwise stated.

[0052] (Term Definition) Hereinafter, in order to make the present invention easier to understand, some technical and scientific terms are specifically defined. Unless specifically defined in this specification, all other technical and scientific terms used in this specification have the ordinary meanings understood by those skilled in the art.

[0053] The three-letter code and one-letter code of amino acids used in the present invention refer to J.biol.chem, 243, p3558 (1968, IUPAC-IUB Commission).

[0054] The "antigen-binding protein" described in the present invention generally refers to a protein containing an antigen-binding portion, and optionally, in the antigen-binding portion, a scaffold or framework portion that enables the use of a three-dimensional structure that promotes the binding of the antigen-binding protein to the antigen. Typically, it may include 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) that 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 the antigen. Examples of antigen-binding proteins include antibodies, antigen-binding fragments (Fab, Fab', Fv fragments, F(ab') 2 , scFv, di-scFv, and / or dAb), immune complexes, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody mimetics, chimeric antigen receptors, or fusion proteins, etc., but are not limited thereto, as long as they exhibit a predetermined antigen-binding activity.

[0055] The "antibody" described in the present invention refers to a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds, that is, an immunoglobulin. Since the amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins are different, their antigenicity is also different. Accordingly, immunoglobulins may be classified into five types, also called immunoglobulin isotypes, namely, IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same type of Ig may be classified into different subclasses depending on the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG may be classified into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ chains or λ chains depending on the constant region. Each of the five types of Ig may have either a κ chain or a λ chain.

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

[0057] The term "complementary determining region (CDR)" refers to one of six hypervariable regions in the variable domain of an antibody that are mainly involved in binding to an antigen. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The 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 rule" (see Kabat et al. (1991)) is used to determine the boundaries of the amino acid sequences of the CDRs.

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

[0059] The terms "monoclonal antibody" and "mAb" refer to an antibody population that is substantially homogeneous, and excluding possible variant antibodies, the individual antibodies constituting the population are the same and / or bind to the same epitope. Generally, unlike a polyclonal antibody preparation that contains different antibodies against different determinants, a monoclonal antibody preparation has each monoclonal antibody against a single determinant on the antigen. Thus, "monoclonal" refers to the property of an antibody obtained from a substantially homogeneous antibody population and is not construed as being produced by any particular method. The monoclonal antibodies described in the present invention may be produced by various techniques known to those skilled in the art, including, but not limited to, the hybridoma method, recombinant DNA method, phage display method, transgenic method, and the like.

[0060] The term "humanized monoclonal antibody" refers to an antibody produced by transplanting the CDR sequences of a mouse into the variable region framework of a human antibody, i.e., into a different type of human germline antibody framework sequence. Since chimeric antibodies carry a large amount of mouse protein components, the induction of heterologous reactions can be overcome. In order to avoid a decrease in activity due to the attenuation of immunogenicity, minimal back mutations may be made to the variable region framework sequence of the human antibody to maintain activity. To produce a humanized antibody, the mouse CDR region may be inserted into the human framework sequence using methods known in the art (see U.S. Patent No. 5,225,539 by Winter, U.S. Patents No. 5,530,101, 5,585,089, 5,693,762, 6,180,370 by Queen et al., and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals that do not produce endogenous immunoglobulins after immunization and can produce a complete human antibody repertoire may be used (e.g., see 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 No. WO02 / 43478). Another method for humanizing an antibody is 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 non-random binding reactions between two molecules, such as the reaction between an antibody and the antigen it targets. The strength or affinity of a specific binding interaction can be represented by the dissociation equilibrium constant (K D ). In this specification, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which represents the binding affinity between an antibody and an 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, an antibody binds with an affinity (K D ) of less than about 10 -8 M, for example, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M or even smaller values.

[0062] The term "identity" generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are the same as those in a second reference polypeptide sequence or a portion thereof, after aligning the sequences, optionally introducing gaps (GAPS), to achieve the maximum percent sequence identity, provided that no conservative substitutions are considered when determining sequence identity. Alignments for determining the percent amino acid / nucleotide sequence identity can be achieved by various methods known in the art, e.g., using generally available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR). One of ordinary skill in the art can determine appropriate parameters for measurement and alignment, including any algorithms necessary to achieve a maximum alignment over the entire length of the sequences being compared. The percent identity may be measured over the entire length of the determined polypeptide / polynucleotide sequence or over a shorter length, e.g., the length of a fragment obtained from the determined longer polypeptide / polynucleotide sequence. It should be understood that the length of any fragment obtained from the sequences shown in the tables, figures, or sequence listings herein may also be used as the length for measuring percent identity. Sequences having "% identity" retain the important biological activity (such as antibody binding specificity) of the sequence to which they are compared or from which they are derived. Sequences having one or more amino acid substitutions, deletions, or additions, or any combination thereof, retain the important biological activity (such as antibody binding specificity) of the sequence to which they are compared or from which they are derived. Nucleotide sequences having "% identity" or nucleotide sequences that differ by three, six, fifteen, thirty, or forty-five nucleotides or less can achieve a function similar to that of the nucleotide sequence to which they are compared or from which they are derived, e.g., the expressed proteins can all 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 change the assumed properties of a protein / polypeptide containing an 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. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain, for example, a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having a similar size, shape, charge, chemical properties such as the ability to form covalent or hydrogen bonds). In the art, families of amino acid residues having similar side chains are defined. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, preferably, the corresponding amino acid residue is replaced with another amino acid residue belonging to the same family having the same side chain. Methods for identifying conservative substitutions of amino acids 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 medium into which a polynucleotide can be inserted. When a protein encoded by the inserted polynucleotide can be expressed in the vector, the vector is called an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection so that the carried genetic material element 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 artificial chromosomes derived from P1 (PACs)), phages (such as λ phage or M13 phage), animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40), etc. The vector may contain various expression control elements including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, reporter genes. Further, the vector may also 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 contains a plasmid or vector comprising a nucleic acid molecule described herein, or can express an antibody or an antigen-binding fragment thereof described herein. The cells may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny cells are not necessarily identical morphologically or genomically to the original parent cell, but may be any that can express the antibody or an antigen-binding fragment thereof described herein. The cells can be obtained by transfecting cells in vitro using a vector described herein. The cells may be prokaryotic cells (e.g., E. coli), or eukaryotic cells (e.g., yeast cells, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some cases, the cells may be mammalian cells. For example, the mammalian cells may be CHOK1 cells.

[0066] The term "pharmaceutically acceptable carrier" refers to a carrier that is well-known in the art and is pharmacologically and / or physiologically compatible with the subject and the active ingredient (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 retardants, 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, for example, Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, etc., but are not limited thereto. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogs. Absorption retardants include, but are not limited to, monostearates, gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as phosphate buffered saline), alcohols, and polyols (such as glycerin). Preservatives include various antibacterial and antifungal agents, such as, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc., but are not limited thereto. Stabilizers have the ordinary meaning understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, and include, but are not limited to, sodium glutamate, gelatin, SPGA, saccharides (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or their degradation products (such as lactalbumin hydrolysate).

[0067] The term "pharmaceutical composition" generally refers to a formulation in which the biological activity of the active ingredient is present in an effective form and which does not contain any other ingredients having unacceptable toxicity to the subject to whom the composition is administered. The composition is sterile.

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

[0069] The term "effective amount" refers to an amount sufficient to obtain a desired effect or a part thereof. For example, an effective amount for preventing a disease (e.g., PRLR-positive related) refers to an amount sufficient to prevent, inhibit, or delay the occurrence of the disease (e.g., PRLR-positive related disease), and an effective amount for treating a disease refers to an amount sufficient to cure or at least inhibit a part of the disease and its complications in a patient suffering from the disease. The measurement of this effective amount is within the ability of those skilled in the art. For example, an amount effective for treatment is determined from the severity of the disease being treated, the overall state of the patient's immune system, the general state of the patient (e.g., age, weight, gender), the mode of drug administration, other treatments being carried out simultaneously, etc.

[0070] The term "chimeric antigen receptor" refers to a chimeric protein obtained by binding the antigen-binding site of an antibody that a chimeric antigen receptor T cell recognizes a specific antigen in vitro to the intracellular portion of the CD3-δ chain or FcεRIγ. By transduction, the patient's T cells are transfected to express the chimeric antigen receptor. As a result, after the patient's T cells are "recoded", a large number of specific CAR-T cells can be generated. When the recoded chimeric antigen receptor T cells are introduced into the patient's body, this chimeric antigen receptor can specifically track and recognize T cells like a GPS and guide the T cells to kill the cells. Most chimeric antigen receptors consist of an extracellular antigen-binding region (composed of a light chain and a heavy chain derived from a monoclonal antibody, and both are connected by a flexible hinge region to form a single-chain antibody), a transmembrane domain, and an intracellular signaling region. The CAR structure is obtained by genetically recombining the scFv that recognizes the relevant antigen and the intracellular signaling domain "immunoreceptor tyrosine activation motif" in vitro.

[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) CDR is the complementarity-determining region in the immunoglobulin variable region. VH is the variable region of the antibody heavy chain. VL is the variable region of the antibody light chain. HC is the antibody heavy chain. LC is the antibody light chain. IgG is immunoglobulin G. AbM is the AbM CDR definition method by Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), and this definition method integrates part of the definitions of both Kabat and Chothia. Kabat is the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). Chothia is the immunoglobulin numbering system proposed by Chothia et al., and it is a classical rule for identifying the boundaries of CDR regions based on the positions of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883). IMGT is the numbering system based on the international ImMunoGeneTics information system (The international ImMunoGeneTics information system (registered trademark) (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 when showing 50% effect or binding. IC 50 is the concentration when showing 50% inhibition. ELISA is enzyme-linked immunosorbent assay. PCR is polymerase chain reaction. HRP is horseradish peroxidase. PRLR is the prolactin receptor. hFc is the Fc region of a human IgG antibody. K D is the dissociation equilibrium constant. HCDR1 is complementarity-determining region 1 in the variable region of the immunoglobulin heavy chain. HCDR2 is complementarity-determining region 2 in the variable region of the immunoglobulin heavy chain. HCDR3 is complementarity-determining region 3 in the variable region of the immunoglobulin heavy chain. LCDR1 is complementarity-determining region 1 in the variable region of the immunoglobulin light chain. LCDR2 is complementarity-determining region 2 in the variable region of the immunoglobulin light chain. LCDR3 is complementarity-determining region 3 in the variable region of the immunoglobulin light chain.

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

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

[0075] In some embodiments, the antigen-binding protein provided by the present invention comprises the following heavy chain variable region VH and light chain variable region VL, namely, (1) A heavy chain variable region VH comprising the amino acid sequence shown by SEQ ID NO: 2, and / or a light chain variable region VL comprising the amino acid sequence shown by 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 compared 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 compared 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 protein provided by the present invention is the following heavy chain HC and light chain LC, namely, (1) A heavy chain HC comprising the amino acid sequence shown by SEQ ID NO: 8, and / or a light chain LC comprising the amino acid sequence shown by SEQ ID NO: 9 or (2) a heavy chain and a light chain, wherein the heavy chain has 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 chain of (1), and / or the light chain has 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, and comprising a heavy chain and a light chain.

[0077] In some specific embodiments, the PRLR antigen-binding protein produced by the present invention comprises the combination of the heavy chain and the light chain described in Table 1 below. [Table 1] Table 1: Sequence characteristics of the PRLR antigen-binding protein JPEG2025517759000002.jpg117147

[0078] (Antibody derivatives and bispecific antibodies) The antigen-binding protein provided by the present invention described above comprises an antibody or an antigen-binding fragment that can be derivatized (e.g., by connecting to another molecule, e.g., another polypeptide or protein). Generally, the derivatization (e.g., labeling) of an antibody or its antigen-binding fragment does not adversely affect its binding to PRLR. Therefore, it is intended that the antibodies or antigen-binding fragments of the present invention also include such derivatized forms. For example, the antibody or its antigen-binding fragment of the present invention can be connected to one or more other molecular groups to form a bispecific antibody, a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., avidin or a polyhistidine tag) that can mediate the binding of an antibody or antigen-binding fragment to another molecule.

[0079] One of the derivatized antibodies is a labeled antibody. For example, the antibody of the present invention or its antigen-binding fragment may be connected to a detectable marker. The detectable marker described in the present invention may be any substance that can be detected by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such markers are well known in the art, and examples thereof include 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 32P), 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 for binding to avidin (e.g., streptavidin) modified by said markers, but not limited thereto. Patents teaching the use of such markers include, but are not limited to, U.S. Patent Nos. 3817837, 3850752, 3939350, 3996345, 4277437, 4275149, 4366241 (all of which are hereby incorporated by reference into this specification). Said detectable markers are detectable by methods known in the art. For example, radioactive markers are detectable by photographic film or scintillation detectors, fluorescent markers are detectable by photodetectors that detect luminescence. Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate, and heat - generating markers are detected simply by visualizing the colored marker. In some embodiments, such markers can be used in immunological detection (e.g., enzyme - linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, said detectable markers may be connected to the antibody or antigen - binding fragment of the present invention via linkers of different lengths to reduce potential steric hindrance.

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

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

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

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

[0084] (Production of Nucleic Acids, Vectors, Host Cells, and Antibodies) The antigen-binding protein of the present invention may be produced by various methods known in the art, such as genetic engineering recombination 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, and then host cells are transfected. Next, the transfected host cells are cultured under specific conditions to express the antibody of the present invention.

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

[0086] In some embodiments, the present invention provides a cloning vector or an expression vector comprising the isolated nucleic acid molecule of the present invention. In some embodiments, the vector is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In some embodiments, the vector can express the antibody of the present invention or its antigen-binding fragment 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., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, 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 CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).

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

[0088] (Pharmaceutical uses, treatment methods, pharmaceutical compositions and administration devices) The present invention provides the use of the PRLR antigen-binding protein, antigen-binding protein derivative, multispecific antibody, immune cell, antibody-drug conjugate in the manufacture of a drug for preventing and / or treating a PRLR blocker, a PRLR-positive disease.

[0089] Accordingly, the present invention provides a method for blocking PRLR and preventing and / or treating a PRLR-positive disease by using the PRLR antigen-binding protein, antigen-binding protein derivative, multispecific antibody, immune cell, antibody-drug conjugate.

[0090] In some embodiments, the present invention provides a therapeutic pharmaceutical composition 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. "Pharmaceutically acceptable carrier" includes, but is not limited to, diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents, binders.

[0091] In some embodiments, the pharmaceutical composition further comprises a combination therapeutic agent, which includes, but is not limited to, chemotherapeutic agents, radiotherapy agents, immunosuppressive agents, cytotoxic drugs.

[0092] In some embodiments, in the pharmaceutical composition, the antigen-binding protein of the present invention and the combination therapeutic agent are provided as separate components or as components of one composition. Thus, the antigen-binding protein of the present invention and the combination therapeutic agent may be combined or administered 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 various routes (e.g., oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, topical, intramedullary, intraventricular). In any particular situation, the optimal route of administration is determined by the specific antibody, the individual, the characteristics and severity of the disease, and the health status of the individual.

[0094] In some embodiments, the present invention also provides an administration device for administering the antigen-binding protein, antigen-binding protein derivative, multispecific antibody, immune cell, antibody-drug conjugate and the pharmaceutical composition comprising the components, and the device (i) An infusion module used for administering a pharmaceutical composition containing an active ingredient to a subject, (ii) The pharmaceutical composition to be infused, which contains 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 pharmacodynamic 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 the injection routes applicable include intravenous, intramuscular, intraarterial, intramembranous, intracystic, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal injection, and bolus administration. In another preferred embodiment, the parenteral administration includes topical, epidermal, or transmucosal administration, for example, intranasal, oral, vaginal, rectal, sublingual, or topical application. In another preferred embodiment, the infusion module is a needleless subcutaneous injection device, a microinfusion pump, a transdermal administration device, a bolus administration device, or an osmotic device.

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

[0097] In one aspect, the present invention provides a kit containing the antigen-binding protein of the present invention. In some embodiments, the antigen-binding protein of the present invention is provided with a detectable marker. In a preferred embodiment, the kit further includes a second antibody that specifically recognizes the antibody of the present invention or its antigen-binding fragment. Preferably, the second antibody further includes a detectable marker.

[0098] In the present invention, the detectable marker may be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, or chemistry. Particularly preferably, such a marker can be used for immunological detection (for example, enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).

[0099] The present invention provides a detection for the expression of PRLR, comprising contacting one or more PRLR antibodies of the present invention (optionally bound to a detectable moiety) with a biological sample (individual cells, tissues, or body fluids), and detecting whether the sample is positive for the expression of PRLR, or detecting whether the expression of the sample has changed (e.g., decreased or increased) compared to a control sample. In some embodiments, the tissue or body fluid is peripheral blood, white blood cells in peripheral blood, biopsy tissue (e.g., lung or skin biopsy tissue), and tissue. The method can be used for diagnostic purposes 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, for example, a cell line or an 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 the antibody or its antigen-binding fragment under conditions that allow the antibody or its antigen-binding fragment described in the present invention 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 a sample derived from the subject with the antibody or its antigen-binding fragment, a multispecific antibody, or an antibody-drug conjugate described in the present invention under conditions that allow the formation of a complex between the antibody or its antigen-binding fragment and PRLR, and detecting the formation of the complex, provided that an increase in the amount of PRLR compared to a healthy control indicates a disease such as a 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 tumors include, but are 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. In a preferred embodiment, the kit includes an instruction manual describing its usage method.

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

[0106] 1.2 Preparation and screening of hybridoma cells Three to four days after the final boost immunization in mice, mouse spleen cells and mouse myeloma cells SP2 / 0 were fused by the PEG method using the conventional hybridoma technique. The fused cells were uniformly mixed in a complete medium (components: RPMI1640 medium, GlutaMax, 1% Penicillin-streptomycin, 1×HAT, 20% FBS (all products were purchased from Gibco)), and dispensed at 3×10 6 cells / 200 μL / well into 30 96-well culture plates and cultured. Seven to twelve days later, the supernatant was obtained, and hybridoma wells with positive human PRLR-ECD binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA). For the hybridoma wells with positive human PRLR-ECD binding, the first and second subclonings were performed by the limiting dilution method to obtain the target hybridoma cell line, named SPGA02-260.

[0107] Here, the method for screening hybridoma wells with positive human PRLR-ECD binding activity by indirect enzyme-linked immunosorbent assay was as follows. The recombinant human PRLR-ECD protein was diluted to 0.5 μg / mL with a coating solution (phosphate buffer, pH 7.4), added to an ELISA plate at 100 μL / well, and coated overnight at 4°C. The plate was washed once with PBST, blocking solution (1% BSA-PBST) was added at 300 μL / well, incubated at 37°C for 2 hours, and then the plate was washed three times with PBST in preparation for use. The collected hybridoma supernatant was 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, HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Abcam, catalog number ab6789) was added, incubated at 37°C for 30 minutes, and then the plate was washed three times with PBST again, tapped on 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, left to stand in the dark at room temperature (20 ± 5°C) for 5 minutes, and then 50 μL of 2M H 2SO 4 The stop solution was added to stop the reaction of the substrate, and the OD value at 450 nm was read using a microplate reader to analyze the binding ability of the tested hybridoma supernatant to the target antigen PRLR-ECD.

[0108] Example 2: Large-scale preparation and identification of mouse anti-human PRLR monoclonal antibody In serum-containing complete medium, the screened hybridoma cell line was amplified, centrifuged and replaced with serum-free medium (SFM), and the cell density was adjusted to 1-2×10 7 / mL, and cultured under the conditions of 5% CO 2 2 for 2 weeks at 37 °C, centrifuged to obtain the culture supernatant, 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 antibody to human PRLR-ECD The binding ability of the mouse antibody to human PRLR was measured by indirect enzyme-linked immunosorbent assay (ELISA). The specific method was as follows. Recombinant human PRLR-ECD protein was diluted to 0.5 μg / mL with coating buffer (phosphate buffer, pH 7.4), added to ELISA plates at 100 μL / well, and coated overnight at 4 °C. The plates were washed once with PBST, blocking buffer (1% BSA-PBST) was added at 300 μL / well, incubated at 37 °C for 2 hours, and then the plates were washed three times with PBST. The purified antibody was diluted to 1 μg / mL with 1% BSA-PBST, serially diluted 1:3, added to ELISA plates at 100 μL / well, and incubated at 37 °C for 1 hour. The plates were washed three times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Abcam, catalog number ab6789) was added, incubated at 37 °C for 30 minutes, and then the plates were washed three times with PBST again, tapped on absorbent paper to remove all residual liquid as much as possible, 100 μL of TMB (purchased from KPL, catalog number 52-00-03) was added to each well, and left to stand in the dark at room temperature (20 ± 5 °C) for 5 minutes. Then, 50 μL of 2M H 2 SO 4 stop solution was added to stop the reaction of the substrate, and 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 the EC 50 50 was 20.9 ng / mL and 0.14 nM.

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

[0112] In this experiment, the 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 into six gradients from 20 μg / mL with a six-fold gradient, and 100 μL of the serially diluted antibody was obtained for each, and 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), and the maximum concentration of the monoclonal antibody used was 10 μg / mL). The cells were washed twice with PBS to remove unbound SPGA02-260. Furthermore, the cells were incubated with 50 μL of anti-mouse fluorescent secondary antibody labeled with 5 μg / mL Alexa Fluor 488 (purchased from Thermo, Invitrogen, catalog number A11001) at 4 °C in the dark for 40 minutes, the cells were washed twice with PBS to remove unbound secondary antibody, and finally the cells were resuspended in 100 μL of PBS, and the binding affinity of SPGA02-260 to the cells was measured with a flow cytometer (purchased from Beckman, model CytoFLEX), and the obtained data was fitted and analyzed with 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 that highly expressed PRLR on the cell surface, and its EC 50 was 26.0 ng / mL, which was 0.17 nM.

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

[0115] The breast cancer cells T47D in the logarithmic growth phase were digested with trypsin and resuspended in complete medium, and seeded in a 6-well plate at 2×10 6 cells / 1 mL / well, and CO 2It was cultured overnight at 37°C in an incubator. The next day, after the cells adhered to the wall, the medium was removed from the 6-well plate, 2 mL of room temperature sterile PBS was gently added to each well to wash away the remaining medium, and then the PBS was removed. In 1640 medium without fetal bovine serum, SPGA02-260 was diluted to concentrations of 20 μg / mL, 2 μg / mL, and 0.2 μg / mL. 1 mL of the antibody with the different concentrations was slowly added to the cells in each well. After culturing in an incubator for 30 minutes, 500 μL / well of a 500 ng / mL PRL (ACRO, Cat#PRN-H5257) solution (prepared in 1640 medium) was slowly added to each well. After addition, the 6-well plate was gently shaken and then placed in the CO 2 incubator for continuous culture. After acting for 30 minutes, the 6-well plate was placed on ice, the medium was removed, and 2 mL of sterile PBS pre-cooled on ice was gently added to each well for washing once. After removing the PBS, 200 μL of RIPA lysis buffer (TEKNOVA, Cat#R3792) was added to each well. After uniform mixing, it was allowed to stand for 10 minutes for lysis. The cell lysate in the plate was collected into a centrifuge tube, centrifuged at 13,000 rpm for 10 minutes to obtain the supernatant, quantified by the BCA method, and then stored in an -80°C refrigerator for subsequent use or the phosphorylation of STST5 (pSTAT5) and phosphorylation of ERK1 / 2 (pERK1 / 2) were measured by the normal Western blot method.

[0116] pSTAT5 was measured as follows. After performing SDS-PAGE electrophoresis (100 μg / lane) on the above cell lysate, it was transferred to a PVDF membrane by electrophoretic transfer, blocked with 1% BSA-TBST (shaken at room temperature for 1 hour), added with 1 μg / mL (diluted with 1% BSA-TBST) of rabbit anti-human phosphorylated STAT5 (Tyr694) (purchased from CST, Cat#94205S), incubated for 2 hours with shaking at room temperature, washed 3 times with TBST, then added with HRP-labeled goat anti-rabbit IgG secondary antibody (purchased from abcam, Cat#ab97051, diluted 10,000-fold with 1% BSA-TBST according to the instruction), incubated for 1 hour with shaking at room temperature, washed 3 times with TBST, then an appropriate amount of Pierce™ ECL Western Blotting Substrate solution (purchased from Thermo, catalog number 32209) was added dropwise to the PVDF membrane, 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. After performing SDS-PAGE electrophoresis (100 μg / lane) on the above cell lysate, it was transferred to a PVDF membrane by electrophoretic transfer, blocked with 1% BSA-TBST (shaken at room temperature for 1 hour), added with 1 μg / mL (diluted with 1% BSA-TBST) rabbit anti-Phospho-p44 / 42 MAPK (Erk1 / 2, Thr202 / Tyr204) Antibody (cell signaling technology, Cat#9101), incubated with shaking at room temperature for 2 hours, washed 3 times with TBST, then added with HRP-labeled goat anti-rabbit IgG secondary antibody (purchased from abcam, Cat#ab97051, diluted 10,000-fold with 1% BSA-TBST according to the instruction manual), incubated with shaking at room temperature for 1 hour, washed 3 times with TBST, then an appropriate amount of Pierce™ ECL Western Blotting Substrate solution (purchased from Thermo, catalog number 32209) was added dropwise to the PVDF membrane, and automatic imaging was performed in a biomolecular imager (purchased from Thermo, model CL1500) at room temperature in the dark.

[0118] β-actin was measured as follows. After performing SDS-PAGE electrophoresis (100 μg / lane) on the above cell lysate, it was transferred to a PVDF membrane by electrophoretic transfer, blocked with 1% BSA-TBST (shaken at room temperature for 1 hour), and 1 μg / mL (diluted with 1% BSA-TBST) of mouse anti-human β-actin monoclonal antibody (purchased from Thermo, Cat#MA1-140) was added. It was incubated for 2 hours with shaking at room temperature, washed 3 times with TBST, and then HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Beijing Bioclone Immunotech Co., Ltd., Cat#BF03001, diluted 10,000-fold with 1% BSA-TBST according to the instructions) was added. It was incubated for 1 hour with shaking at room temperature, washed 3 times with TBST, and then an appropriate amount of Pierce(™) ECL Western Blotting Substrate solution (purchased from Thermo, catalog number 32209) was dropped onto the PVDF membrane, and autoradiography was automatically performed in a biomolecular imager (purchased from Thermo, model CL1500) at room temperature in the dark.

[0119] The results are shown in Figure 3. In each of the measured samples, the content of β-actin as a control was consistent. During the experiment, in T47D cells, STAT5 was not phosphorylated when there was no stimulatory effect of PRL, and STAT5 was clearly phosphorylated when there was the action of PRL. The SPGA02-260 antibody could completely inhibit the phosphorylation of STAT5 induced by PRL stimulation at concentrations of 20 μg / mL and 2 μg / mL, and could still partially inhibit the phosphorylation of STAT5 at a concentration of 0.2 μg / mL.

[0120] The results are shown in Figure 4. In each of the measured samples, the content of β-actin as a control was consistent. During the experiment, when there was no stimulating effect of PRL in T47D cells, ERK1 / 2 was not phosphorylated. When there was the effect of PRL, ERK1 / 2 was clearly phosphorylated. SPGA02-260 at concentrations of 20 μg / mL, 2 μg / mL, and 0.2 μg / mL could completely inhibit the phosphorylation of ERK1 / 2 induced by PRL stimulation.

[0121] Example 6: Endocytosis of Mouse Antibody by Target Cell T47D In this example, the endocytosis of SPGA02-260 in human breast cancer cell T47D was measured by the Fluorescence activated Cell Sorting (FACS) method.

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

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

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

[0125] JPEG2025517759000003.jpg40124

[0126] Specific information on the related sequences is shown in Table 1 above. However, The full length of the SPGA02-260 heavy chain variable region gene sequence was 360 bp, encoding 120 amino acid residues, the amino acid sequence was as described in SEQ ID NO: 2, and the nucleotide sequence was as described in SEQ ID NO: 3. The full length of the light chain variable region gene sequence was 321 bp, encoding 107 amino acid residues, the amino acid sequence was as described in SEQ ID NO: 4, and the nucleotide sequence was as described in SEQ ID NO: 5.

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

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

[0129] The experimental method referred to Example 3.

[0130] The results are shown in Figure 6. It was revealed that SPGA02-ch260 binds to PRLR with high affinity, and the EC 50 was 12.9 ng / mL, which was 0.09 nM.

[0131] Example 9: Measurement of the binding affinity of a chimeric antibody 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 referred to Example 4.

[0133] The results are shown in Figure 7. It was revealed that SPGA02-ch260 binds to T47D cells highly expressing PRLR with high affinity, and the EC 50 was 31.0 ng / mL, which was 0.21 nM.

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

[0135] Mouse PRLR protein (purchased from Sino Biological Inc., catalog number 50457-M08H), that is, mouse PRLR, was used to coat a 96-well ELISA plate at 0.5 μg / well, and the cross-reactivity of SPGA02-ch18 and SPGA02-ch260 with mouse PRLR was measured. The remaining part of the specific experimental method referred to Example 8.

[0136] The results are shown in Figure 8. It was revealed that SPGA02-ch260 can bind to mouse PRLR protein, and the EC 50 was 297.3 ng / mL, which was significantly lower than the binding ability to human PRLR (EC 50 was 12.9 ng / mL).

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

[0138] To determine the epitope that binds to human PRLR of SPHA02-260, the extracellular domain (PRLR-ECD) gene of human PRLR was obtained by searching the literature and NCBI (amino acids at positions 1 to 234 of NP_000940.1 registered in NCBI, the amino acid sequence is described in SEQ ID NO: 1, provided that the amino acid sequence of fibronectin type-III 1 (27 to 128), which is a functional domain, is described in SEQ ID NO: 10, and the amino acid sequence of fibronectin type-III 2 (129 to 229), which is a functional domain, is described in SEQ ID NO: 11). Fc fusion proteins of two functional domains of PRLR-ECD, namely fibronectin type-III 1 and fibronectin type-III 2, were expressed respectively.

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

[0140] The results are shown in Figure 9. SPGA02-260 could specifically bind to reduced and denatured human PRLR, and it was detected that SPGA02-260 bound to PRLR at the second functional domain of human PRLR-ECD, namely Fibronectin type-III 2.

[0141] Human PRLR-ECD-Fibronectin type-III 2 was degraded to obtain 10 polypeptides that partially overlapped with each other, and the amino acid sequences of each polypeptide were as follows. 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] By using the ELISA method and measuring the binding status of SPGA02-260 to the above 10 polypeptides, the epitope that binds to the PRLR of SPGA02-260 was determined.

[0143] The results are shown in Figure 10. Since SPGA02-260 binds only to the above polypeptide 7, that is, the polypeptide consisting of amino acids at positions 189 to 208 at the N-terminus of the human PRLR protein, it was found 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 chain variable region and the heavy chain variable region of the candidate mouse antibody SPGA02-260 were analyzed, and three antigen - complementary 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 complementary determining regions are HCDR1: TVSGFSLTRNGV (SEQ ID NO: 12), HCDR2: IWGDGST (SEQ ID NO: 13), HCDR3: AKEGLYYYGRYFDV (SEQ ID NO: 14), and the amino acid sequences of the light chain complementary determining regions are LCDR1: KASQDVGSAV (SEQ ID NO: 15), LCDR2: WASTRHT (SEQ ID NO: 16), LCDR3: QQYSNYPLT (SEQ ID NO: 17).

[0145] From the germline database, a humanized template that most matched each of the above mouse antibody FR regions was selected. Next, the CDR regions of the mouse antibody were transplanted into the selected humanized template to replace the CDR regions of the human template, thereby forming a humanized antibody heavy chain variable region (SPGA02 - hu260VH, SEQ ID NO: 18), which was recombined with the human IgG1 constant region (including the S228P mutation, SEQ ID NO: 6) to form a humanized monoclonal antibody heavy chain (SPGA02 - hu260H, SEQ ID NO: 20). A humanized light chain (SPGA02 - hu260L, SEQ ID NO: 21) was formed by the recombination of the humanized antibody light chain variable region (SPGA02 - hu260VL, SEQ ID NO: 19) and the human kappa chain constant region. The heavy chain and light chain of the humanized antibody were respectively constructed into the pcDNA3.4 expression vector, transfected into Expi - 293F cells, and purified with Protein A to obtain the humanized antibody SPGA02 - hu260. It was found by SDS - PAGE electrophoresis and SEC - HPLC that each SPGA02 - 260 antibody had the correct molecular weight and a purity exceeding 95%.

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

[0147] The experimental method referred to Example 3.

[0148] The results are shown in Figure 11. It was revealed that SPGA02-hu260 binds to PRLR with high affinity, and the EC 50 was 8.98 ng / mL and 0.06 nM.

[0149] Example 14: Inhibition of humanized antibody SPGA02-hu260 against signal transduction caused by the binding of PRLR to PRL The experimental method referred to Example 5. The working concentration of the antibody SPGA02-hu260 was 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 as a control was consistent.

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

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

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

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

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

[0156] In T47D, ERK1 / 2 was clearly phosphorylated by the action of GH1. The SPGA02-hu260 antibody could still clearly inhibit the phosphorylation of ERK1 / 2 induced by GH1 stimulation even when the concentration decreased to 0.2 μg / mL.

[0157] Example 16: Inhibition of the proliferation of BAF3-huPRLR cells by the antibody SPGA02-hu260 Mouse B cells BAF3 in the logarithmic growth phase were cultured in RPMI 1640 culture medium (complete medium) containing 10 ng / mL of mouse IL3 (Beyotime Biotech Inc, P5912) and 10% FBS (Gibico), and the cells were infected with a lentivirus (Jiman Biotechnology (Shanghai) Co., Ltd, catalog number GM-10162OL01) that overexpresses human PRLR, and screening and subcloning were performed in the presence of 5 μg / mL of puromycin dihydrochloride (Jiman Biotechnology (Shanghai) Co., Ltd, catalog number GM-040401-1) to obtain a human PRLR stable overexpression cell line, namely, BAF3-huPRLR.

[0158] BAF3-huPRLR cells were cultured in RPMI 1640 cell culture medium containing 40 ng / mL of human PRL (ACRO Biosystems Co., Ltd., PRR-H52Ha), 2×10 5Inoculate into a 96-well cell culture plate at a density of cells / 100 μL. After diluting the antibody SPGA02-hu260 to 30 μg / mL with 1640 culture medium without PRL, serially dilute it 10-fold into 8 gradients, and then add 100 μL / well to the 96-well cell culture plate containing the BAF3-huPRLR cells. Incubate the cells in a CO 2 incubator for 72 hours. Then, add CCK8 reagent according to the instructions, incubate at 37 °C for 2 hours, and then measure OD 450 .

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

[0160] Several of the above-described examples merely illustrate some embodiments of the present invention. Although they are described specifically and in detail, they should not be construed as limitations on the scope of the present invention patent. Those skilled in the art may make some modifications and improvements on the premise of not departing from the idea of the present invention, and all of these belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the scope of the claims set forth hereinafter.

Claims

1. The following complementarity-determining regions, namely, (1) Heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 12 or a variant sequence thereof (2) Heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence shown in SEQ ID NO: 13 or a variant sequence thereof (3) Heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence shown in SEQ ID NO: 14 or a variant sequence thereof (4) Light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence shown in SEQ ID NO: 15 or a variant sequence thereof (5) Light chain complementarity-determining region 2 (LCDR2) comprising the amino acid sequence shown in SEQ ID NO: 16 or a variant sequence thereof (6) Light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 17 or a variant sequence thereof, a PRLR antigen-binding protein, characterized by comprising the same.

2. The antigen-binding protein, wherein (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) 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 compared to VH of (1), and / or 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 compared to VL of (1) or (3) VH having one or more amino acid substitutions, deletions or additions or any combination thereof compared to VH of (1), and / or VL having one or more amino acid substitutions, deletions or additions or any combination thereof compared to VL of (1), the substitution being a conservative substitution, the antigen-binding protein according to claim 1.

3. The antigen-binding protein further comprises a human IgG1 constant region and a human κ (kappa) chain constant region, 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 κ (kappa) chain constant region is as set forth in SEQ ID NO: 7, the VH of the PRLR antigen-binding protein is connected to the human IgG1 constant region to form a heavy chain, and the VL of the PRLR antigen-binding protein is connected to the human κ (kappa) chain constant region to form a light chain. The antigen-binding protein according to claim 2.

4. The 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) a heavy chain and a light chain, wherein compared with the heavy chain and light chain of (1), the heavy chain has 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 chain has 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. The antigen-binding protein according to claim 3, characterized by comprising a heavy chain and a light chain.

5. (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 compared 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 compared 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, and characterized in that it is a humanized PRLR antigen-binding protein **Claim 6** The humanized PRLR antigen-binding protein is (1) a heavy chain HC containing the amino acid sequence shown in SEQ ID NO: 20, and / or a light chain LC containing the amino acid sequence shown in SEQ ID NO: 21 or (2) a heavy chain and a light chain, wherein the heavy chain has 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 chain of (1), and / or the light chain has 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 light chain of (1), and characterized in that it is the humanized PRLR antigen-binding protein according to claim 5 **Claim 7** A nucleic acid molecule encoding the PRLR antigen-binding protein according to any one of claims 1 to 4 or the humanized PRLR antigen-binding protein according to claim 5 or 6 **Claim 8** A vector comprising the nucleic acid molecule according to claim 7.

9. A host cell comprising the nucleic acid molecule according to claim 7 or the vector according to claim 8.

10. A pharmaceutical composition comprising the PRLR antigen-binding protein according to any one of claims 1 to 4, the humanized PRLR antigen-binding protein according to claim 5 or 6, the nucleic acid molecule according to claim 7, the vector according to claim 8, and / or the host cell according to claim 9.

11. Use of the PRLR antigen-binding protein according to any one of claims 1 to 4, the humanized PRLR antigen-binding protein according to claim 5 or 6, the nucleic acid molecule according to claim 7, the vector according to claim 8, the host cell according to claim 9, and / or the pharmaceutical composition according to claim 10 in the manufacture of a drug, kit and / or administration device for preventing and / or treating PRLR-positive diseases.

12. Use according to claim 11, characterized in that the PRLR-positive disease is a tumor or alopecia.

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