Prolactin receptor antibodies and their applications

Abstract

The present invention relates to a prolactin receptor antibody and its applications, and more specifically to an antibody or its antigen-binding fragment that targets PRLR, or a variant that retains its PRLR-binding activity while having at least 85% sequence identity with the antibody or its antigen-binding fragment, wherein the antibody targets an extracellular segment that targets the prolactin receptor. The antibody of the present invention binds to PRLR with high affinity.

Description

Technical Field

[0001] The present invention relates to the fields of biology and medicine; more specifically, the present invention relates to an antibody that specifically binds to a prolactin receptor and its applications.

Background Art

[0002] The prolactin receptor (PRLR) is a single-pass transmembrane protein and a type I cytokine receptor, and is a homologous protein with interleukin 2, 3, 4, 6, 7, erythropoietin, granulocyte macrophage colony-stimulating factor receptor, etc. Prolactin, the natural ligand of the prolactin receptor, is a polypeptide hormone consisting of 199 amino acids, and is mainly secreted by prolactin-secreting cells in the human pituitary gland. After prolactin binds to the prolactin receptor in the homodimeric form, it induces a conformational change in the receptor dimer, activates Jak2 bound to the intracellular segment of the receptor, and Jak2 catalyzes the formation of an active dimer of phosphorylated Stat5. The Stat5 phosphorylated dimer enters the cell nucleus and regulates downstream gene expression. In addition to the main Jak2-Stat5 signaling pathway, activation of the prolactin receptor activates Src, promotes cell proliferation, or causes activation of MARK, etc. Also, growth factor hormones and placental lactogen can bind to and activate the prolactin receptor. Therefore, activation of PRLR mediates diverse biological functions and can be used as a potential target for disease intervention in certain benign diseases and malignant tumors.

[0003] The normal physiological functions mediated by the prolactin receptor include promoting cell growth, differentiation, development, and lactation. Human prolactin receptor cDNA was initially isolated from liver and breast cancer libraries. The mature protein has 598 amino acids in full length, and due to different mRNA splicing, there are different subtypes of the prolactin receptor: L-type, I-type, S1a-type, and S1b-type. These subtypes share a common extracellular segment and have intracellular segments of different lengths. The prolactin receptor is considered a potential target for breast and prostate cancer treatment, although its role in the development and progression of breast cancer remains debated. In contrast, PRL, synthesized outside the pituitary gland autocrinely and / or paracrinely, is thought to be involved in prostate tumorigenesis. In mouse models, overexpression of PRL in the prostate can lead to benign prostatic hyperplasia, neoplastic lesions in situ, and even prostatic adenoma. From an epidemiological perspective, the expression of PRL and phosphorylated Stat5 in patient prostate tissue is positively correlated with tumor malignancy and the degree of disease invasion.

[0004] Prolactin receptors are not only targets for potential malignancies but also important targets in various benign lesions. Clinical studies have shown that enhanced PRLR-mediated signaling is associated with the gynecological disease endometriosis. Exploratory clinical studies have shown that in women diagnosed with endometriosis and hyperprolactinemia, administration of the dopamine agonist quinagolide (a prolactin synthesis regulator) for more than four months significantly reduced the size of peritoneal endometriotic lesions. Furthermore, prolactin interferes with the hair follicle cycle, and elevated blood prolactin levels are associated with hair loss. Prolactin receptors have been shown to be expressed in hair follicles, and prolactin has been shown to induce the degenerative phase of the hair follicle cycle. In mouse models, dopamine receptor agonists (PRL inhibitors) can promote hair growth in mice. In summary, PRLR is a promising pharmacological target, and antibodies or antigen-binding fragments targeting PRLR have applicability to a variety of PRLR-related diseases, offering broad market prospects. [Overview of the project]

[0005] The present invention first provides an antibody or its antigen-binding fragment that targets PRLR, or a variant that retains its PRLR-binding activity while having at least 85% sequence identity with the antibody or its antigen-binding fragment, wherein the antibody targets an extracellular segment that targets the prolactin receptor. Preferably, the extracellular segment comprises amino acids at positions 1-210 of SEQ ID NO: 99 or 101.

[0006] In one or more embodiments, the antibody comprises three HCDRs in the heavy chain variable region indicated by any one of SEQ ID NO: 1-23, and / or three LCDRs in the light chain variable region indicated by any one of SEQ ID NO: 24-41. Preferably, the antibody comprises three HCDRs in the heavy chain variable region indicated by any one of SEQ ID NO: 3, 5, 16, 20-23, and / or three LCDRs in the light chain variable region indicated by any one of SEQ ID NO: 26, 28, 36, 40, 41.

[0007] In one or more embodiments, the HCDR1 of the antibody comprises one selected from: SEQ ID NO: 42-52, or a sequence having at least 85% sequence identity thereto.

[0008] In one or more embodiments, the HCDR2 of the antibody comprises one selected from: SEQ ID NO: 53-66, or a sequence having at least 85% sequence identity thereto.

[0009] In one or more embodiments, the HCDR3 of the antibody comprises one selected from: SEQ ID NO: 67-75, or a sequence having at least 85% sequence identity thereto.

[0010] In one or more embodiments, the LCDR1 of the antibody comprises one selected from: SEQ ID NO: 76-83, or a sequence having at least 85% sequence identity thereto.

[0011] In one or more embodiments, the LCDR2 of the antibody comprises one selected from: SEQ ID NO: 84-90, or a sequence having at least 85% sequence identity thereto.

[0012] In one or more embodiments, the LCDR3 of the antibody comprises one selected from: SEQ ID NO: 91-98, or a sequence having at least 85% sequence identity thereto.

[0013] In one or more embodiments, the antibody comprises HCDR1, HCDR2, HCDR3 of the antibodies shown in any one row of Table 1, or a sequence having at least 85% sequence identity thereto. In one or more embodiments, the antibody comprises LCDR1, LCDR2 and LCDR3 of the antibodies shown in any one row of Table 1, or a sequence having at least 85% sequence identity thereto.

[0014] In one or more embodiments, the HCDR1, HCDR2, and HCDR3 of the antibody are selected from any one of the following groups or sequences having at least 85% sequence identity with them: (1) SEQ ID NO:43, SEQ ID NO:55, and SEQ ID NO:67, (2) SEQ ID NO:43, SEQ ID NO:66, and SEQ ID NO:67, (3) SEQ ID NO:44, SEQ ID NO:56 and SEQ ID NO:68, (4) SEQ ID NO: 50, SEQ ID NO: 63 and SEQ ID NO: 73, and / or The LCDR1, LCDR2, and LCDR3 of the aforementioned antibody are selected from any one of the following groups or sequences having at least 85% sequence identity with them: (1) SEQ ID NO:77, SEQ ID NO:84 and SEQ ID NO:91, (2) SEQ ID NO: 82, SEQ ID NO: 90 and SEQ ID NO: 97.

[0015] In one or more embodiments, the antibody includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antibodies shown in any one row of Table 1.

[0016] In one or more embodiments, the light chain variable region of the antibody includes a mouse or human light chain FR region. In one or more embodiments, the heavy chain variable region of the antibody includes a mouse or human heavy chain FR region.

[0017] In one or more embodiments, the heavy chain variable regions FR1, FR2, FR3, and FR4 of the antibody are each independently selected from the heavy chain variable regions FR1, FR2, FR3, and FR4 shown in any one of SEQ ID NO: 1-23, and / or the light chain variable regions FR1, FR2, FR3, and FR4 of the antibody are each independently selected from the light chain variable regions FR1, FR2, FR3, and FR4 shown in any one of SEQ ID NO: 24-41.

[0018] In one or more embodiments, the FR region of the heavy chain variable region of the antibody is selected from the FR regions of the heavy chain variable region shown in any one of SEQ ID NO: 3, 5, 16, 20-23.

[0019] In one or more embodiments, the FR region of the light chain variable region of the antibody is selected from the FR regions of the light chain variable region shown in any one of SEQ ID NO: 26, 28, 36, 40, or 41.

[0020] In one or more embodiments, the antibody comprises a VH, a VL, or VH and VL of an antibody shown in any row of Table 1, or a sequence having at least 85% sequence identity thereto.

[0021] In one or more embodiments, the VH of the antibody comprises a sequence shown in any one of SEQ ID NO: 3, 5, 16, 20 - 23 or a sequence having at least 85% sequence identity thereto. Alternatively or additionally, the VL of the antibody comprises a sequence shown in any one of SEQ ID NO: 26, 28, 36, 40, 41 or a sequence having at least 85% sequence identity thereto.

[0022] In one or more embodiments, for the antibody, VH is shown in SEQ ID NO: 3 and VL is shown in SEQ ID NO: 26, VH is shown in SEQ ID NO: 5 and VL is shown in SEQ ID NO: 28, VH is shown in SEQ ID NO: 16 and VL is shown in SEQ ID NO: 36, VH is shown in SEQ ID NO: 20 or 21 and VL is shown in SEQ ID NO: 40, VH is shown in SEQ ID NO: 22 and VL is shown in SEQ ID NO: 40, or VH is shown in SEQ ID NO: 23 and VL is shown in SEQ ID NO: 41.

[0023] In one or more embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0024] In one or more embodiments, the antibody is a multispecific antibody, preferably a bispecific antibody.

[0025] In one or more embodiments, the antibody is a monoclonal antibody.

[0026] In one or more embodiments, the antibody is a chimeric antibody or a fully human antibody.

[0027] The present invention further provides a fusion protein or antibody complex comprising an antibody or its antigen-binding fragment as described herein.

[0028] The present invention provides polynucleotides selected from the following: (1) The coding sequence of an antibody or its antigen-binding fragment, fusion protein, or antibody complex as described in any one embodiment herein; (2) Complementary sequence of (1).

[0029] The present invention further provides a nucleic acid construct that expresses an antibody or its antigen-binding fragment, fusion protein, or antibody complex described in any one embodiment herein, or a polynucleotide described in any one embodiment herein.

[0030] In one or more embodiments, the nucleic acid construct is a vector, such as an integration vector, a cloning vector, or an expression vector.

[0031] The present invention provides a phage comprising an antibody or its antigen-binding fragment as described in any one embodiment herein, or a library comprising such phage.

[0032] In one or more embodiments, the antibody or its antigen-binding fragment is displayed on the surface of the phage.

[0033] The present invention further, (1) Express and / or secrete an antibody or antigen-binding fragment described in any one embodiment of this specification; (2) containing polynucleotides as described herein; and / or (3) comprising nucleic acid constructs described herein Provides host cells.

[0034] In one or more embodiments, the host cell is selected from prokaryotic cells or eukaryotic cells.

[0035] In one or more embodiments, the host cell is a mammalian cell.

[0036] The present invention further provides a method for producing an antibody or its antigen-binding fragment, comprising culturing host cells described herein under conditions suitable for producing the antibody or its antigen-binding fragment, and optionally purifying the antibody or its antigen-binding fragment from the culture.

[0037] The present invention further provides pharmaceutical compositions comprising antibodies or their antigen-binding fragments, fusion proteins, antibody complexes, polynucleotides, nucleic acid constructs, phages or host cells, and pharmaceutically acceptable auxiliary materials as described herein.

[0038] In one or more embodiments, the auxiliary raw material is a carrier, a diluent, or an excipient.

[0039] In one or more embodiments, the pharmaceutical composition is used for the treatment of cancer.

[0040] In one or more embodiments, the cancer is a cancer associated with PRLR. Preferably, the cancer is selected from ovarian cancer, melanoma, prostate cancer, colorectal cancer, stomach cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain tumor, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies. Hematological malignancies are selected from myeloma, chronic leukemia, and acute leukemia.

[0041] The present invention further provides the application of antibodies or their antigen-binding fragments, fusion proteins, antibody complexes, polynucleotides, nucleic acid constructs, or host cells described in any one embodiment herein in the preparation of pharmaceuticals for preventing or treating diseases.

[0042] In one or more embodiments, the disease includes cancer, endometriosis, alopecia, osteoporosis, obesity, and benign lesions caused by PRLR activation.

[0043] In one or more embodiments, the cancer is a cancer associated with PRLR. Preferably, the cancer is selected from ovarian cancer, melanoma, prostate cancer, colorectal cancer, stomach cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain tumor, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies. Hematological malignancies are selected from myeloma, chronic leukemia, and acute leukemia.

[0044] The present invention further provides a method for inhibiting the growth of tumor cells in a test subject, inhibiting benign lesions by PRL activating PRLR, or treating or preventing a disease, the method comprising administering a therapeutically effective amount of an antibody or its antigen-binding fragment, fusion protein, antibody complex, or pharmaceutical composition described in any one embodiment of the present invention to a patient in need.

[0045] In one or more embodiments, the disease includes cancer, endometriosis, alopecia, osteoporosis, obesity, and benign lesions caused by PRLR activation.

[0046] In one or more embodiments, the cancer is a cancer associated with PRLR. Preferably, the cancer is selected from ovarian cancer, melanoma, prostate cancer, colorectal cancer, stomach cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain tumor, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies. Hematological malignancies are selected from myeloma, chronic leukemia, and acute leukemia.

[0047] The present invention further provides a kit for detecting PRLR used in evaluating the therapeutic effect of a drug or in diagnosing cancer, the kit comprising an antibody or its antigen-binding fragment, fusion protein, antibody complex, polynucleotide, nucleic acid construct, phage, or host cell as described in any one embodiment herein.

[0048] In one or more embodiments, the kit further includes a reagent for detecting the binding of PRLR to an antibody or its antigen-binding fragment, fusion protein, or antibody complex. For example, the reagent is one that detects the binding by, for example, an enzyme-linked immunoassay.

[0049] In one or more embodiments, the reagent for detecting the binding is a detectable marker, such as biotin, that can bind to an antibody or its antigen-binding fragment, a fusion protein, or an antibody complex. The detectable marker is either bound to the antibody or its antigen-binding fragment, or present separately in the kit.

[0050] The present invention further provides a non-diagnostic method for detecting the presence of PRLR in a sample, the method comprising: incubating the sample with an antibody or its antigen-binding fragment, fusion protein, or antibody complex described in any one embodiment herein; and determining the presence of PRLR in the sample by detecting the binding of PRLR to the antibody or its antigen-binding fragment, fusion protein, or antibody complex. The detection is by enzyme-linked immunoassay.

[0051] The present invention further provides applications of antibodies or antigen-binding fragments, fusion proteins, or antibody complexes described in any one embodiment herein in the manufacture of kits used for detecting PRLR in a sample, evaluating the therapeutic effect of a drug, or diagnosing cancer. [Brief explanation of the drawing]

[0052] Figure 1 shows immunofluorescence detection of PRLR endocytosis mediated by the PRLR humanized antibody published here. Figure 2 shows the detection of PRLR-positive cell line toxicity mediated by the PRLR humanized antibody of this disclosure. [Modes for carrying out the invention]

[0053] Unless otherwise defined, the implementation of this invention utilizes conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the technical scope of the present art. These techniques are described in detail in publications such as "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (edited by MJ Gait, 1984), "Animal Cell Culture" (edited by RIFreshney, 1987), "Methods in Enzymology" (Academic Press, Inc.), "Current Protocols in Molecular Biology" (edited by FMAusubel et al., 1987 edition and regularly updated editions), "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994), "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988), and "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0054] The prolactin receptor (PRLR) is a single-pass transmembrane protein, a type I cytokine receptor, and homologous to interleukin 2, 3, 4, 6, 7, erythropoietin, and granulocyte-macrophage colony-stimulating factor receptor (CRT). The PRLR binds to human prolactin, growth factor hormones, and placental lactogens, mediating various biological functions such as cell proliferation, differentiation, and lactation. Abnormal activation of the PRLR is associated with various human diseases, including tumors, endometriosis, alopecia, hyperprolactinemia, and osteoporosis.

[0055] This invention utilizes the extracellular segment of the PRLR protein as an epitope peptide to obtain anti-human PRLR hybridoma monoclonal antibodies. Subsequently, a series of PRLR-specific antibodies were obtained by screening the antibody gene library using phage display technology and performing humanization modifications. Then, antibodies with high affinity, high specificity, and high functional activity were identified using methods such as ELISA, affinity testing, receptor internalization, blocking tests, and target cell killing. These antibodies or their antigen-binding fragments have good safety and targetability and can specifically bind to the extracellular segment of the PRLR protein.

[0056] antibody In this specification, the term “antibody” includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions having polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments (particularly antigen-binding fragments, e.g., Fab, F(ab′)2, and Fv). In this specification, “antibody” and “immunoglobulin” are used interchangeably.

[0057] Conventional "antibodies" are heterotetrameric glycoproteins that contain a basic four-chain antibody unit, consisting of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for μ and ε isotypes), and a hinge region (Hinge) located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by another constant domain (CL) at its other end. The paired VH and VL together form the antigen-binding site. For the structure and properties of different categories of antibodies, refer to Basic and Clinical Immunology, 8th Edition (edited by Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6). Light chains from any vertebrate species are classified into two distinct types, called κ and λ, based on the amino acid sequence of their constant domain. Based on relatively small differences in CH sequence and function, the γ and α classes are further classified into subclasses; for example, in humans, the following subclasses are expressed: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2. The antibody heavy chain may further include a heavy chain constant region, which may contain the constant region or variant sequences thereof of human or mouse-derived IgG1, 2, 3, or 4. The antibody light chain may further include a light chain constant region, which includes a human or mouse-derived κ or λ chain constant region or a variant thereof.

[0058] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain. The variable domains of the heavy and light chains are called "VH" and "VL," respectively. These domains are usually the most variable parts of the antibody (compared to other antibodies of the same type) and contain the antigen-binding site.

[0059] The term "variable" refers to a situation where a specific segment of a variable domain differs significantly in the antibody sequence. Variable domains mediate antigen binding and limit the specificity of a particular antibody against a specific antigen. However, variability is not evenly distributed across all amino acids spanning the variable domain. Rather, it is concentrated in three segments called highly variable regions (HVRs) (present in the variable domains of both the light and heavy chains): HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain, and LCDR1, LCDR2, and LCDR3 in the light chain variable domain, respectively. The more highly conserved portions within the variable domain are called framework regions (FRs). The natural heavy and light chain variable domains each contain four FR regions (FR1, FR2, FR3, and FR4), which are mostly linked by forming a cyclic structure in a β-sheet conformation, and in some cases, three HVRs that form part of the β-sheet structure are linked. The HVR of each chain is held very tightly by the FR region and, together with the HVR of other chains, contributes to the formation of the antibody's antigen-binding site. Typically, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domain does not directly participate in antibody-antigen binding but exhibits diverse effector functions, such as the antibody's involvement in antibody-dependent cell-mediated cytotoxicity. Antibodies have various variable region labeling schemes, including Chothia, Kabat, IMGT, and Contact. In this specification, the IMGT labeling scheme is used exemplarily.

[0060] The "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of the antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In the isotypes of IgG, IgA, and IgD antibodies, the Fc region consists of two identical protein fragments: the CH2 domain and the CH3 domain of the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. While the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the Fc region of the human IgG heavy chain is usually defined as the sequence fragment from the amino residue at position C226 or P230 of the heavy chain to the carboxyl terminus, although this number is based on the EU index, as in Kabat. As used herein, the Fc region may be a natural sequence Fc or a variant Fc.

[0061] An "antigen-binding fragment" typically refers to a part of an antibody molecule containing amino acids responsible for specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an "epitope," as mentioned earlier. As previously stated, the antigen-binding domain may typically include the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH), but not necessarily both. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, and Fv fragments; disulfide-linked Fv; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment whose half-life can be extended by chemical modification or incorporation into liposomes. An Fd fragment, for example, has two VH regions and typically retains some of the antigen-binding function of the complete antigen-binding domain. Examples of antibody antigen-binding fragments include: (1) Fab fragments, which are monovalent fragments having VL, VH, constant light chain (CL), and CH1 domains; (2) F(ab')2 fragments, which are bivalent fragments having two Fab fragments linked by disulfide crosslinks in the hinge region; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains in a single antibody arm; (5) dAb fragments having a VH domain (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which is incorporated herein by reference); (6) isolated complementarity-determining regions (CDRs); and (7) single-stranded Fv (scFv), e.g., derived from scFV libraries.The two domains of the Fv fragment, VL and VH, are encoded by independent genes but are linked together by a synthetic linker in a recombinant manner. The synthetic linker is prepared so that the VL and VH regions pair up to form a single monovalent protein chain (called single-chain Fv (scFv)) (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). These antibody fragments are obtained using conventional techniques known to those skilled in the art and are evaluated for function in the same manner as complete antibodies.

[0062] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly non-covalently bound heavy chain variable domain and a light chain variable domain. From the folding of these two domains, six highly variable loops (three in the heavy chain and three in the light chain) protrude, providing antigen-binding amino acid residues and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) can recognize and bind to an antigen, although its affinity is lower than that of a complete binding site. "Single-chain Fv" can also be abbreviated as "scFv" or "scFv," and is an antibody fragment in which the VH and VL domains of an antibody are linked to a single polypeptide chain. Preferably, the sFv polypeptide further contains a polypeptide linker between the VH and VL domains, so that the sFv forms the desired antigen-binding structure. In the case of heavy chain antibodies or nanoantibodies, scFv is VHH.

[0063] In this specification, the term “monoclonal antibody” means an antibody obtained from a substantially homogeneous group of antibodies, i.e., each antibody constituting the population is identical except for mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts and may occur naturally. Monoclonal antibodies are highly specific and target a single antigenic site. Compared to polyclonal antibody preparations (which typically contain different antibodies against different determinants (epitopes)), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, an advantage of monoclonal antibodies is that they are cultured and synthesized by hybridomas and are not contaminated by other immunoglobulins. The modifier “monoclonal” is a characteristic of antibodies obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring antibody production by a specific method. For example, the monoclonal antibodies used in the present invention can be produced by a variety of techniques, such as hybridoma generation, phage display, recombinant DNA generation, and single-cell sequencing, as well as techniques for generating human or human-like antibodies from animals having a partial or complete human immunoglobulin locus or a gene encoding a human immunoglobulin sequence.

[0064] In this specification, monoclonal antibodies include "chimeric" antibodies, which are those in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence from an antibody of a particular species or belonging to a particular antibody category or subcategory, and the rest of the chain is identical or homologous to a corresponding sequence from an antibody of a different species or belonging to a different antibody category or subcategory, as well as fragments of such antibodies that possess the desired biological activity.

[0065] In this disclosure, the term “mouse-derived antibody” means a mouse-derived monoclonal antibody that binds to human PRLR, prepared based on the knowledge and skills of the art. During preparation, the PRLR antigen is injected into the test subject to isolate the antibody having the desired sequence or functional properties. In one preferred embodiment of this disclosure, the mouse PRLR antibody or its antigen-binding fragment may further include the light chain constant region of the mouse κ, λ chain or its mutant sequence, or further include the heavy chain constant region of the mouse IgG1, IgG2, IgG3 or its mutant sequence.

[0066] The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse-derived antibody with the constant region of a human antibody, which can mitigate the immune response caused by mouse-derived antibodies. To construct a chimeric antibody, first, a hybridoma secreting a specific monoclonal antibody derived from a mouse is created. Then, the variable region gene is cloned from the mouse hybridoma cells, and if necessary, the constant region gene of a human antibody is cloned. Finally, the mouse variable region gene and the human constant region gene are combined to create a chimeric gene, which is then inserted into a vector, and the chimeric antibody molecule is expressed in eukaryotic or prokaryotic cells. In a preferred embodiment of the present invention, the antibody light chain of the chimeric antibody further includes a light chain constant region of a human κ, λ chain or a mutant sequence thereof. The antibody heavy chain of the chimeric antibody further includes a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or a mutant sequence thereof, preferably a human IgG1 heavy chain constant region.

[0067] The "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains at least a minimum number of sequences derived from non-human immunoglobulins. Therefore, a "humanized antibody" usually refers to a non-human antibody in which the variable domain framework region has been replaced with sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody (excluding the CDR) is encoded by human-derived polynucleotides, or is identical to this antibody (excluding the CDR). The CDR (partially or entirely encoded by nucleic acids derived from non-human organisms) is transplanted into the β-sheet backbone of the human antibody variable region to produce an antibody with specificity determined by the transplanted CDR. The term "humanized antibody" is also called a CDR-grafted antibody. Methods for producing this type of antibody are well known in this field, and are produced, for example, using mice with a genetically modified immune system. Human species antibody variable region framework (FR) sequences can be obtained from the ImMunoGeneTics (IMGT) website at http: / / imgt.cines.fr. The common sequences of human-derived antibodies can be obtained from the website https: / / plueckthun.bioc.uzh.ch / antibody / Modelling / HuCAL / index.html (J.Mol.Biol. 296, 57-86 (2000)). To avoid a decrease in immunogenicity and simultaneously prevent a decrease in antibody activity, activity can be maintained by performing reverse mutations (revert mutations) on the variable region of the human antibody. In this invention, the antibody includes a humanized variant.

[0068] "Human antibody" means an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced from a human, and / or an antibody produced using any of the techniques disclosed herein for producing human antibodies. The definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries.

[0069] In this disclosure, the term "binding to PRLR" means being able to interact with human PRLR.

[0070] The term "antigen-binding epitope" as defined in this publication refers to a discontinuity on an antigen that is recognized by the antibody or antigen-binding fragment as defined in this publication, and is a site in three-dimensional space.

[0071] In some embodiments, the present invention further provides nanoantibodies, heavy-chain antibodies, antibodies, or antigen-binding fragments thereof that compete with the antigen-binding region of any antibody of the present invention to bind to the same epitope on the PRLR, i.e., nanoantibodies, heavy-chain antibodies, antibodies, or antigen-binding fragments thereof that can compete with each other for binding to the antigen-binding region of any antibody of the present invention and to the PRLR.

[0072] In specific embodiments of the present invention, the anti-PRLR antibody comprises the CDR group shown in any row of Table 1:

[0073] [Table 1]

[0074] The FR1, FR2, FR3, and FR4 of the antibody described herein are each independently selected from the VH or VL FR1, FR2, FR3, and FR4 shown in any row of Table 1. Preferably, in one or more embodiments, the FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody are each independently selected from the FR1, FR2, FR3, and FR4 of the heavy chain variable region shown in any one of SEQ ID NO: 1-23; the FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody are each independently selected from the FR1, FR2, FR3, and FR4 of the light chain variable region shown in any one of SEQ ID NO: 24-41.

[0075] In one or more embodiments, the antibody comprises VH, VL, or VH and VL of the antibody shown in any row of Table 1. Preferably, VH is shown in SEQ ID NO:3 and VL is shown in SEQ ID NO:26, or VH is shown in SEQ ID NO:5 and VL is shown in SEQ ID NO:28, or VH is shown in SEQ ID NO:16 and VL is shown in SEQ ID NO:36, or VH is shown in SEQ ID NO:20 or 21 and VL is shown in SEQ ID NO:40, or VH is shown in SEQ ID NO:22 and VL is shown in SEQ ID NO:40, or VH is shown in SEQ ID NO:23 and VL is shown in SEQ ID NO:41.

[0076] In one or more embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region. Exemplarily, the heavy chain of the antibody comprises the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or a sequence having at least 85% sequence identity thereto; the light chain of the antibody comprises the light chain constant region of human κ chain, λ chain or a sequence having at least 85% sequence identity thereto. Preferably, the heavy chain of the antibody comprises the heavy chain constant region of human IgG1 or a sequence having at least 85% sequence identity thereto; the light chain of the antibody comprises the light chain constant region of human κ chain or a sequence having at least 85% sequence identity thereto.

[0077] In this specification, the antibody may be a multispecific antibody comprising one, two, or more chains or antigen-binding fragments as described herein. The multispecificity may be against PRLR and another antigen, or against two different epitopes of PRLR.

[0078] The present invention further includes derivatives and analogues of the antibody. “Derivatives” and “analogues” refer to polypeptides that possess essentially the same biological function or activity as the antibody of the present invention. Derivatives or analogues of the present invention may be (i) polypeptides having substituents on one or more amino acid residues, or (ii) polypeptides obtained by fusing a mature polypeptide with another compound (e.g., a compound that extends the polypeptide half-life, e.g., polyethylene glycol), or (iii) polypeptides obtained by fusing an added amino acid sequence to this polypeptide sequence (e.g., a lead sequence or secretion sequence or a sequence for purifying this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these derivatives and analogues are well known to those skilled in the art.

[0079] The “mutation” in the “mutant sequence” described in this disclosure includes, but is not limited to, “reverse mutations,” “conservative modifications,” or “conservative substitutions or replacements.” “Conservative modifications” or “conservative substitutions or replacements” as described in this disclosure refer to the substitution of an amino acid in a protein with another amino acid having similar properties (e.g., charge, side chain size, hydrophobic / hydrophilicity, back chain conformation and rigidity), and such modifications can be made frequently without altering the biological activity of the protein. Those skilled in the art generally recognize that the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, the likelihood of inhibiting biological activity by substitutions of structurally or functionally similar amino acids is low.

[0080] Provided that the antibody activity is not substantially affected, those skilled in the art can obtain variants of the antibody or its functional fragment sequence by modifying one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) in the sequence of the antibody of the present invention. These variants include, but are not limited to, one or more (generally 1–50, preferably 1–30, more preferably 1–20, most preferably 1–10) amino acid deletions, insertions, and / or substitutions, and the addition of one or more (generally 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acids to the C-terminus and / or N-terminus. In the art, conservative substitutions by amino acids with similar properties generally do not alter the function of the protein. For example, substitutions are made in the FR region and / or Fc region by amino acids with similar properties. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Furthermore, the addition of one or more amino acids to, for example, the C-terminus and / or N-terminus typically does not alter the function of the protein. All of these are considered to fall within the scope of protection of this invention.

[0081] The antibody variant forms described herein include homologous sequences, conserved variants, allelic variants, native mutants, induced mutants, and proteins encoded by DNA hybridizable with the DNA encoding the antibody of the present invention under high-stringency or low-stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention. In some embodiments, the sequences of the variants described in the present invention may have at least 95%, 96%, 97%, 98%, or 99% identity with their originating sequences. The sequence identity described in the present invention can be measured using sequence analysis software. For example, the computer program BLAST, particularly BLASTP or TBLASTN, is used with default parameters. The present invention also includes molecules having an antibody heavy chain variable region having a CDR, insofar as its CDR has 90% or more (preferably 95% or more, most preferably 98% or more) homology with the CDRs identified herein.

[0082] The antibodies of the present invention can be prepared using conventional methods in the art, such as hybridoma technology and phage display technology. Alternatively, the antibodies of the present invention may be expressed in other cell lines. Suitable mammalian host cells can be transformed with the sequence encoding the antibodies of the present invention, and then the host cells can be cultured to purify the antibodies. Transformation can be carried out by any known method, for example, including packaging polynucleotides in a virus (or viral vector) and transducing the host cells with the virus (or vector). The transformation program used is determined by the host being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and microinjection of DNA directly into the nucleus. Suitable mammalian cell lines as hosts include, but are not limited to, various immortalized cell lines well known in this field and obtained from the American Center for Typical Cell Cultures (ATCC), as well as, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc.

[0083] This disclosure further provides a fusion protein of the PRLR extracellular region and the mouse antibody IgG2a Fc fragment. The fusion protein is a protein product obtained by co-expressing two genes obtained by DNA recombination. The recombinant PRLR extracellular region Fc fusion protein is a fusion protein obtained by co-expressing the PRLR extracellular region and the mouse antibody IgG2a Fc fragment through DNA recombination. The aforementioned PRLR extracellular region refers to the portion of the PRLR protein that is expressed outside the cell membrane.

[0084] The present invention further comprises a fusion protein containing the anti-PRLR antibody described herein and other polypeptides. In some embodiments, the other polypeptide is located at the N-terminus and / or C-terminus of the antibody. In some embodiments, the other polypeptide includes polypeptides that localize the antibody to different organelles, tags for purification or immunoreaction, transmembrane proteins or their transmembrane regions, chimeric antigen receptors or their components (such as extracellular domains, hinge regions, transmembrane regions, signaling domains, and costimulatory domains).

[0085] The present invention further comprises an antibody conjugate containing the anti-PRLR antibody described herein, wherein a bioactive cytotoxic agent is conjugated to the antibody by chemical bonding, and the antibody acts as a carrier to target and deliver the cytotoxic agent to target cells, thereby exerting its effect. Those skilled in the art can select a pharmaceutical product conjugated with the anti-PRLR antibody described herein as needed and in practice.

[0086] nucleic acid The present invention further provides polynucleotides encoding antibodies as described herein. The polynucleotides of the present invention may be in DNA form or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may consist of a coding strand and a non-coding strand. The present invention also includes degenerate variants of polynucleotides encoding fusion proteins, i.e., nucleotide sequences that encode the same amino acid sequence but have a different nucleotide sequence. The RNA may be mRNA that expresses antibodies in vivo and / or in vitro.

[0087] Accordingly, the present invention also relates to a polynucleotide that hybridizes with the aforementioned polynucleotide sequence and has at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to a polynucleotide that can hybridize with the polynucleotide of the present invention under stringent conditions. As used herein, “stringent conditions” means (1) hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 × SSC, 0.1% SDS, 60°C; or (2) addition of a denaturant during hybridization, e.g., 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the two sequences are at least 90%, preferably 95% or more, more preferably 95% identical. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has similar biological functions and activities to mature polypeptides.

[0088] The full-length nucleotide sequence or fragments of the antibody of the present invention can usually be obtained by PCR amplification, recombination, or artificial synthesis. One effective method, especially when the fragment length is short, is to synthesize the sequence in question by artificial synthesis. Typically, several small fragments are first synthesized, and then they are ligated to obtain a longer sequence fragment. Alternatively, a heavy chain coding sequence may be fused with an expression tag (e.g., 6His) to form a fusion protein. After obtaining the sequences of each part of the fusion protein as described above, they can be ligated to obtain the full-length fusion protein.

[0089] Once the relevant sequence is obtained, a large quantity of the relevant sequence can be obtained using a recombination method. Typically, this is cloned into a vector, transfected into cells, and then the relevant sequence is isolated from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) related to the present invention include biomolecules that exist in isolated forms. Currently, the DNA sequence encoding the protein of the present invention (or its fragments, or derivatives thereof) can be obtained by complete chemical synthesis. Subsequently, this DNA sequence may be introduced into cells with various existing DNA molecules (or vectors) known in the art. Alternatively, mutations may be introduced into the protein sequence of the present invention by chemical synthesis. Each part of the fusion protein can be cloned sequentially into a vector, or it can be cloned after being integrated as a full-length fusion protein.

[0090] The present invention also relates to nucleic acid constructs, which include the polynucleotides described herein and one or more regulatory sequences operably linked to these sequences, such as regulatory sequences suitable for expressing DNA or RNA as antibodies in vivo or in vitro. The polynucleotide sequences of the present invention can be manipulated in various ways to ensure the expression of the antibody. The nucleic acid construct may be manipulated before insertion into the vector, depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0091] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically operably ligated to the sequence encoding the protein to be expressed. The promoter may be any nucleotide sequence exhibiting transcriptional activity in a selected host cell, including mutant, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. An example of a suitable promoter is the early cytomegalovirus (CMV) promoter sequence, a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably ligated to it. Another example of a suitable promoter is elongation factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but are not limited to, the Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-chain terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EBV very early promoter, Rous sarcoma virus promoter, and human gene promoters (such as actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter). Furthermore, the use of inducible promoters can also be considered. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence that operably binds to the inducible promoter when expression is desired and turn it off when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0092] The regulatory sequence may be a suitable transcriptional terminator sequence, which is recognized by the host cell and terminates transcription. The terminator sequence is operably ligated to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator functional in a selected host cell can be used in this invention. The regulatory sequence may be a suitable read sequence, which is an untranslated region of mRNA important for translation by the host cell. The read sequence is operably ligated to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator functional in a selected host cell can be used in this invention.

[0093] Other regulatory sequences suitable for expressing DNA or RNA in or out of the body are common knowledge in this field.

[0094] In some embodiments, the nucleic acid construct is a vector, such as a cloning vector, expression vector, or integration vector. Expression of the polynucleotide sequence of the present invention is achieved by operably linking the polynucleotide sequence of the present invention to an expression vector. A typical cloning vector includes a transcription and translation terminator, an initiation sequence, and a promoter that control the expression of the desired nucleic acid sequence. An integration vector includes components that integrate the target sequence into the cellular genome. These vectors are used to transform a suitable host cell so that it can express a protein. The vector typically contains sequences used for plasmid maintenance and the cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to as “flanking sequences” in some embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and receptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding the antibody to be expressed, and an optional marker element. An example of an expression vector is, for example, pcDNA3.1.

[0095] Furthermore, there are no restrictions on the type of vector; for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids may be used, and can be modified depending on the host cell to be introduced. Viral vector technology is well known in this art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other manuals on virology and molecular biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0096] To evaluate the expression of a polypeptide or a portion thereof, the vector introduced into cells may also include either or both a selection marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a cell population intended for transfection or infection via the viral vector.

[0097] cell Suitable host cells for introducing the nucleic acid constructs described herein may be prokaryotic cells (e.g., bacterial cells), lower eukaryotic cells (e.g., yeast cells), or higher eukaryotic cells (e.g., mammalian cells). Typical examples include bacterial cells of Escherichia coli, Streptomyces; Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells. Examples of mammalian cells include immune cells, more preferably immune effector cells. "Immune effector cells" are immune cells capable of exerting immune effector functions and include T cells, NK cells, peripheral blood mononuclear cells (PBMCs), neutrophils, eosinophils, and hematopoietic stem cells. Suitable T cells for use in the present invention may be various types of T cells from various sources.

[0098] Methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and such vectors can be introduced into cells by physical, chemical, or biological means. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used being well known in the art. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging. In some embodiments, transduced or transfected immunoeffector cells are grown in vitro after the introduction of nucleic acids or vectors.

[0099] The resulting transformants are cultured in a conventional manner to express the antibody encoded by the gene of the present invention. Depending on the host cells used, the culture medium can be selected from a variety of conventional media. The cells are cultured under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature change or chemical induction), and the cells are re-cultured for a period of time.

[0100] The polypeptides in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant proteins may be separated and purified by various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional refolding, treatment with protein precipitants (salting-out methods), centrifugation, cell disruption by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and other liquid chromatography techniques and combinations thereof. For example, the antibody can be passed through a Protein A or G Sepharose FF column, washed to remove nonspecific binding components, eluted with an acidic buffer, the antibody fragment detected by SDS-PAGE, and the collected antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods such as gel filtration chromatography or ion exchange. The resulting product should be immediately frozen (e.g., at 80°C) or lyophilized.

[0101] Applications and Methods By constructing a nanoantibody library, the inventors screened nanoantibodies and their variants that can bind to PRLR. The binding ability of these antibodies to antigens was verified by protein-level binding detection, affinity detection, competitive inhibition experiments, and tissue cross-reactivity.

[0102] Any antibodies, coding sequences, nucleic acid constructs, and any aspect of cells described herein may be used in the manufacture of pharmaceuticals for the prevention or treatment of various medical conditions and diseases described herein, which are medical conditions or conditions related to PRLR expression, and which refer to diseases directly or indirectly caused by abnormal PRLR expression, usually caused by PRLR overexpression, such as cancer, including but not limited to acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma, such as relapsed or refractory acute lymphoblastic leukemia (r / r ALL), relapsed or refractory diffuse large B-cell lymphoma (r / r DLBCL), and relapsed or refractory follicular lymphoma (r / r FL).

[0103] This specification provides pharmaceutical compositions comprising any one or more antibodies or antigen-binding fragments thereof, fusion proteins, nucleic acid molecules, nucleic acid constructs and cells, and pharmaceutically acceptable auxiliary materials.

[0104] The antibodies, nucleic acids, or cells of the present invention may be administered alone or in combination with diluents and / or other components, such as relevant cytokines or cell populations, as part of a pharmaceutical composition. Here, the pharmaceutical composition may be prepared in the form of a lyophilized or aqueous solution by mixing an active agent of desired purity with any pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is nontoxic to the recipient at the dose and concentration used and may contain at least one of the following: buffers (e.g., neutral buffered saline, sulfate-buffered saline), antioxidants, preservatives, isotonic agents, stabilizers, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), or surfactants. Furthermore, the pharmaceutical composition must be sterile for use in internal administration. The pharmaceutical composition can be sterilized by filtration through a sterile filtration membrane.

[0105] In some embodiments, the pharmaceutical composition may include at least one additive from among cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, growth inhibitors, and active agents necessary for the specific indication to be treated. The specific amount of the additive can be adjusted according to the actual needs.

[0106] The pharmaceutical compositions of the present invention can be administered in an “immunologically effective dose,” “antiotumor effective dose,” “tumor-suppressing effective dose,” or “therapeutic dose.” “Therapy” means that the treatment plan described herein is carried out on a subject to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor size, a decrease in the rate of cancer cell invasion into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). When referring to an “immunologically effective dose,” “antiotumor effective dose,” “tumor-suppressing effective dose,” or “therapeutic dose,” the exact amount of the composition of the present invention to be administered is determined by a physician, taking into account the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and individual differences in the disease. Typically, the T cell-containing pharmaceutical compositions described herein can be administered in doses of 10⁴ to 10⁹ cells / kg body weight, preferably 10⁵ to 10⁶ cells / kg body weight. The T cell compositions can also be administered multiple times in these doses. Cells can be administered in immunotherapy using known infusion techniques (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment plan for a specific patient can be easily determined by medical professionals by monitoring the patient's condition and adjusting the treatment accordingly.

[0107] The compositions can be administered by any convenient method, including by spray, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients by subcutaneous, intradermal, intratumoral, intranodular, intraspinal, intramuscular, intravenous, or intraperitoneal injection. The compositions can be injected directly into tumors, lymph nodes, or infected sites.

[0108] In some embodiments of the present invention, the compositions of the present invention may be combined with other therapies known in the art, including, but not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, they may be used in combination with radiotherapy or chemotherapeutic agents known in the art for the treatment of PRLR-mediated diseases.

[0109] In this specification, "antitumor effect" refers to biological effects such as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an extension of life expectancy, and improvement of various physiological symptoms associated with cancer.

[0110] The terms "patient," "subject," and "individual" are used interchangeably within this text and refer to living organisms, such as mammals, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and their genetically modified species.

[0111] The present invention will be described in further detail below with reference to experimental examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Accordingly, the present invention should not be construed as being limited to the following embodiments, but rather as including any modifications that become apparent as a result of the teachings provided herein. The methods and reagents used in the examples are conventional methods and reagents in the art unless otherwise specified.

[0112] Diagnostics, detection and kits Because the antibody of the present invention has a high affinity for PRLR, it can be used, for example, in binding assays to detect and / or quantify PRLR expressed in tissues and cells. The antibody can be used in studies to further investigate the role of PRLR in disease. The method for detecting PRLR is roughly as follows: obtain a cell and / or tissue sample; detect the level of PRLR in the sample.

[0113] The PRLR antibody of the present invention is used for diagnostic purposes and can be used for the detection, diagnosis, or monitoring of PRLR-related diseases and / or conditions. The present invention provides a method for detecting the presence of PRLR in a sample using classical immunohistochemical methods known to those skilled in the art. PRLR detection can be performed in vivo or in vitro. Examples of suitable methods for detecting the presence of PRLR include ELISA, FACS, and RIA.

[0114] For diagnostic applications, antibodies are typically marked with detectable marker groups. Suitable marker groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanum-based phosphors), enzyme marker groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotin groups, or peptide epitopes intended to be recognized by secondary reports (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Various methods used to mark proteins are already known in the art and can be used in the present invention.

[0115] Another aspect of the present invention provides a method for detecting the presence of a test molecule that competitively binds to PRLR with the antibody of the present invention. An example of one measurement relates to detecting the amount of free antibody in a solution containing a certain amount of PRLR, in the presence or absence of the test molecule. An increase in the amount of free antibody (i.e., antibody not bound to PRLR) indicates that the test molecule can compete with that antibody for binding to PRLR. In one embodiment, the antibody is macbed with a marker group, or with the test molecule, to monitor the amount of free test molecule in the presence and absence of the antibody.

[0116] The present invention further provides a detection kit for detecting PRLR levels, the kit comprising a PRLR antibody, a lysis medium for dissolving a sample, general-purpose reagents and buffers necessary for detection, such as various buffers, a detection label, a detection substrate, etc. The detection kit may also be an in vitro diagnostic device.

[0117] Some specific embodiments 1. An antibody or its antigen-binding fragment that targets PRLR, or a variant that has at least 85% sequence identity with the antibody or its antigen-binding fragment while retaining its PRLR-binding activity, wherein the antibody targets an extracellular segment that targets the prolactin receptor; Preferably, the extracellular segment contains amino acids at positions 1-210 of SEQ ID NO:99 or 101; More preferably, the antibody comprises three HCDRs in the heavy chain variable region shown in any one of SEQ ID NO: 1-23, and / or three LCDRs in the light chain variable region shown in any one of SEQ ID NO: 24-41.

[0118] 2. The HCDR1 of the antibody comprises one of the following selected from: SEQ ID NO: 42-52, or a sequence having at least 85% sequence identity thereto; and / or The HCDR2 of the antibody comprises one of the following selected from: SEQ ID NO: 53-66, or a sequence having at least 85% sequence identity thereto; and / or The HCDR3 of the antibody comprises one of the following selected from: SEQ ID NO: 67-75, or a sequence having at least 85% sequence identity thereto; and / or The LCDR1 of the antibody comprises one of the following selected from: SEQ ID NO: 76-83, or a sequence having at least 85% sequence identity thereto; and / or The LCDR2 of the antibody comprises one of the following selected from: SEQ ID NO: 84-90, or a sequence having at least 85% sequence identity thereto; and / or The LCDR3 of the antibody comprises one of the following: SEQ ID NO: 91-98, or a sequence having at least 85% sequence identity thereto; Preferably, the antibody comprises HCDR1, HCDR2, and HCDR3 of the antibodies shown in any one row of Table 1, or a sequence having at least 85% sequence identity thereto; and / or, the antibody comprises LCDR1, LCDR2, and LCDR3 of the antibodies shown in any one row of Table 1, or a sequence having at least 85% sequence identity thereto; More preferably, the antibody includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antibodies shown in any one row of Table 1. An antibody or its antigen-binding fragment as described in item 1, characterized by the above.

[0119] 3. The light chain variable region of the antibody includes a mouse or human light chain FR region, and the heavy chain variable region of the antibody includes a mouse or human heavy chain FR region; Preferably, the heavy chain variable regions FR1, FR2, FR3, and FR4 of the antibody are each independently selected from the heavy chain variable regions FR1, FR2, FR3, and FR4 shown in any one of SEQ ID NO: 1-23, and / or the light chain variable regions FR1, FR2, FR3, and FR4 of the antibody are each independently selected from the light chain variable regions FR1, FR2, FR3, and FR4 shown in any one of SEQ ID NO: 24-41; More preferably, the antibody includes VH, VL, or VH and VL of the antibody shown in any one row of Table 1, or a sequence having at least 85% sequence identity thereto. An antibody or its antigen-binding fragment as described in item 1 or 2, characterized by the above.

[0120] 4. The antibody further comprises a heavy chain constant region and / or a light chain constant region; and / or The antibody is a multispecific antibody; and / or The antibody is a monoclonal antibody; and / or The aforementioned antibody is either a chimeric antibody or a fully human antibody. An antibody or its antigen-binding fragment as described in item 1 or 2, characterized by the above.

[0121] 5. A fusion protein or antibody complex containing an antibody or its antigen-binding fragment as described in any one of items 1-4.

[0122] 6. (1) An antibody or its antigen-binding fragment as described in any one of items 1-4, or the coding sequence of a fusion protein or antibody complex as described in item 5; (2) Complementary sequence of (1) A polynucleotide selected from among them.

[0123] 7. Expressing an antibody or its antigen-binding fragment as described in any one of items 1-4, or being a fusion protein or antibody complex as described in item 5, or containing a polynucleotide as described in item 6; Preferably, the nucleic acid construct is a vector. Nucleic acid construct.

[0124] 8. A phage containing an antibody or antigen-binding fragment described in any one of items 1-4, or a library containing such phage.

[0125] 9. (1) Express and / or secrete an antibody or antigen-binding fragment described in any one of items 1-4; (2) containing polynucleotides as described in item 6; and / or (3) Containing nucleic acid constructs as described in item 7; A host cell characterized by the following: Preferably, the host cell is a mammalian cell.

[0126] 10. A method for producing an antibody or an antigen-binding fragment, comprising culturing host cells as described in item 9 under conditions suitable for producing the antibody or its antigen-binding fragment, and optionally purifying the antibody or its antigen-binding fragment from the culture.

[0127] 11. A pharmaceutical composition comprising an antibody or its antigen-binding fragment, fusion protein, antibody complex, polynucleotide, nucleic acid construct, phage or host cell, and pharmaceutically acceptable auxiliary material as described in any one of items 1-4.

[0128] 12. Application of an antibody or its antigen-binding fragment as described in any one of items 1-4, a fusion protein or antibody complex as described in item 5, a polynucleotide as described in item 6, a nucleic acid construct as described in item 7, or a host cell as described in item 9 in the preparation of a medicine for the prevention or treatment of a disease; Preferably, the disease includes cancer, endometriosis, alopecia, osteoporosis, obesity, and benign lesions due to PRLR activation; More preferably, the cancer is a cancer associated with PRLR; More preferably, the cancer is selected from ovarian cancer, melanoma, prostate cancer, colorectal cancer, stomach cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain tumor, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies.

[0129] 13. A kit for detecting PRLR used to evaluate the therapeutic effect of a drug or to diagnose cancer, comprising an antibody or antigen-binding fragment described in any one of items 1-4, a fusion protein or antibody complex described in item 5, a polynucleotide described in item 6, a nucleic acid construct described in item 7, a phage described in item 8, or a host cell described in item 9; Preferably, the kit further includes reagents for detecting the binding of PRLR to an antibody or its antigen-binding fragment, fusion protein, or antibody complex.

[0130] 14. Application of an antibody or antigen-binding fragment described in any one of items 1-4, or a fusion protein or antibody complex described in item 5, a polynucleotide described in item 6, a nucleic acid construct described in item 7, a phage described in item 8, or a host cell described in item 9, in the detection of PRLR in a sample, evaluation of the therapeutic effect of a drug, or in the manufacture of a kit used in a cancer diagnostic kit.

[0131] The present invention will be described in further detail below with reference to experimental examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Accordingly, the present invention should not be construed as being limited to the following embodiments, but rather as including any modifications that become apparent as a result of the teachings provided herein. The methods and reagents used in the examples are conventional methods and reagents in the art unless otherwise specified.

[0132] Examples The present disclosure will be further illustrated below with examples, but these examples are not intended to limit the scope of the disclosure. Experimental methods in the examples published herefore, where specific conditions are not described, are typically performed under standard conditions such as those found in Cold Spring Harbor's Antibody Technology Manual and Molecular Cloning Manual, or under experimental conditions recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are standard commercially available reagents.

[0133] Example 1. Preparation of PRLR antigen and detection protein Using the extracellular domain (amino acid sequence at positions 25-234) of the human PRLR protein (Uniprot ID: P16471) or the extracellular domain (amino acid sequence at positions 25-234) of the cynomolgus monkey PRLR protein (Uniprot ID: G8F5S4) as templates, we designed antigen and detection proteins with different fusion tags, including the avi-(his)6 tag (amino acids at positions 211-231 of SEQ ID NO: 99) and the mouse IgG2a Fc(mFc) tag (amino acids at positions 214-446 of SEQ ID NO: 100). The extracellular segment of the human PRLR protein tagged with avi-(his)6 (hPRLR-ECD-avi-his) is shown at SEQ ID NO: 99. The extracellular segment of the human PRLR protein tagged with mouse IgG2a Fc(mFc) (hPRLR-ECD-mFc) is shown at SEQ ID NO: 100. The extracellular segment of the PRLR protein from cynomolgus monkeys tagged with avi-(his)6 (cynoPRLR-ECD-avi-his) is shown as SEQ ID NO:101.

[0134] The DNA sequence encoding the aforementioned protein (Shanghai Biotechnology) was synthesized and inserted into a pcDNA3.1 expression vector to construct a plasmid expressing the recombinant protein. The target plasmid was transfected into Expi293F (Thermo) cells using PEI (polyscience) to express the protein. After 4 days, the supernatant of the cell culture medium was collected, centrifuged at high speed, and the supernatant was collected again and filtered through a 0.22 μm filter to remove any remaining cell debris.

[0135] Antigen proteins were purified using nickel columns or Protein A columns (GE) depending on the type of tag attached. The purified samples were concentrated, identified by SDS-PAGE and mass spectrometry, then packaged and frozen at -80°C for use.

[0136] Example 2. Construction of a PRLR-expressing cell line DNA encoding the complete human PRLR sequence (Uniprot ID: P16471, 1-622) and also containing a sequence encoding the P2A-GFP tag (sequences 625-882 of SEQ ID NO: 102) was synthesized (the encoding amino acid sequence is shown in SEQ ID NO: 102) and inserted into a pcDNA3.1 expression vector. The purified plasmid was cleaved with Pvu I (NEB) enzyme and transfected into HEK293, Cos7, or CHO K1 cells using Lipofectamine LTX (Thermo). 24 hours after transfection, 700-1000 μg / mL of G418 was added and screening was performed. After continuous culture in a G418-containing culture medium for two weeks, GFP expression was detected by flow cytometry, and GFP-positive cells were selected. After single-cell selection and amplification culture, stable transfected cell lines expressing PRLR, HEK293-PRLR, CHOK1-PRLR, and Cos7-PRLR, were constructed.

[0137] Example 3. Acquisition and preparation of anti-human PRLR hybridoma monoclonal antibody 1. Mouse Immunology Six-to-eight-week-old Balb / c female mice (Guangdong Yaokang Biotechnology Co., Ltd.) were selected and immunized with recombinantly expressed protein antigens hPRLR-ECD-avi-his or hPRLR-ECD-mFc (protein concentration: 1-2 mg / mL) mixed thoroughly with an equal volume of adjuvant [selectable options: complete Freund's adjuvant (Sigma, F5881-10ML), incomplete Freund's adjuvant (344291-10ML), TiterMax Gold adjuvant (Sigma, T2684), Imject™ Alum (Thermo, 77161), etc.]. For the initial immunization, an antigen emulsification prepared with complete Freund's adjuvant or TiterMax Gold adjuvant was used, and 50-100 μg of antigen was administered to each mouse by subcutaneous injection. Booster immunizations were performed on days 14 and 28 after the initial immunization. For booster immunizations, an antigen suspension was prepared by mixing protein with incomplete Freund's adjuvant or Imject® Alum. Each mouse was administered 25–50 μg of antigen by intraperitoneal or subcutaneous injection. Blood was collected on day 35 to test antibody titers, and based on the level of antibody titer, it was decided whether to continue booster immunizations or perform a final sprint immunization. Sprint immunizations involved administering 50–100 μg of pure protein solution (protein dissolved in phosphate buffer or physiological saline) by tail vein injection. Three days post-immunization, the mice were euthanized, and samples of their blood, spleen, and lymph nodes were collected for testing and hybridoma preparation. 2. Hybridoma preparation Hybridoma cells were prepared by a method well known to those skilled in the art. Specifically, fresh mouse spleen cells or lymph node cells were prepared as a single-cell suspension. Erythrocyte lysate (Beyotime, C3702) was added and the suspension was treated at room temperature for 5 minutes to lyse the erythrocytes. After centrifugation and counting, the suspension was mixed with an Sp2 / 0 myeloma cell line (with a spleen cell:Sp2 / 0 cell ratio of 2:1), thoroughly washed with 20 mL of electrofusion buffer (BTX cytofusion medium C), and then electrofusion (BTX ECM2001) was performed to prepare hybridoma cells. Using a 9 mL fusion pool, the electrofusion parameters were set as follows (AC parameters: 75V / 75V / 30s / 1.0MHz; DC parameters: 800V / 40μs / 1 / 0.000s; AC parameters: 75V / 75V / 30s / 1.0MHz); 7.2 mL of well-mixed cell suspension was added to the fusion pool, fusion was started immediately, and the fused hybridoma cells were resuspended in HAT complete medium (RPMI 1640 medium containing 10% FBS, 1×HAT, 1×P / S, 1×Glutamax, and 1×Pyruvate) and placed in a 96-well cell culture plate (1×10 5 The cells were inoculated into 100 μl / well and cultured at 37°C in 5% CO2. The culture medium was changed every 3 days. On day 14 after fusion, ELISA testing was performed based on the cell growth status. 3. Screening of hybridoma cells Antigen protein at 3 ug / mL (100 μL / well) was added to a 96-well high-adsorption plate (Nunc MaxiSorp®, 44-2404-21) and incubated overnight at 4°C. The following day, the 96-well plate (300 μL / well) was blocked with PBS blocking solution containing 3% skim milk powder and left at room temperature for 1 hour. The 96-well plate was then washed three times with PBS rinse solution containing 0.1% Tween-20, and 100 μL of hybridoma supernatant was added to each well and left at room temperature for 1 hour. The 96-well plate was washed three times, and 100 μL of 2000-fold diluted HRP-marked goat anti-mouse antibody (Thermo, A10521) was added to each well and incubated at room temperature for 1 hour. After washing the plate four times with washing solution, 50 μL of TMB chromogenic solution (Invitrogen, 002023) was added and allowed to develop for 2-5 minutes. The reaction was stopped by adding 1M sulfuric acid, and the absorbance at 450 nm was recorded using a microplate reader. Positive hybridomas were sorted by flow cytometry, and 1 cell / well was inoculated into a new 96-well plate, cultured, grown, and cryopreserved.

[0138] Example 4. Construction and screening of a phage display library of anti-human PRLR antibodies. RNA from immunized mouse spleen cells was extracted, cDNA was synthesized, and a single-chain antibody (scFv)-phage display library was constructed. Target antibodies were then screened using phage surface display technology. Specifically, after synthesizing cDNA, PCR amplification was performed using degenerate primers, such as the primer combination provided by A. Krebber et al. in Journal of Immunological Methods 201 (1997) 35-55, to obtain the heavy and light chain sequence DNA of the target antibody, and a single-chain antibody-phage display immunolibrary was constructed. Target cloning sequences were obtained by various selection methods well known to those skilled in the art.

[0139] Example 5. Variable region sequences of the heavy and light chains of anti-PRLR antibody Table 2 shows the sequence numbers corresponding to the amino acid sequences of the heavy chain, light chain variable region, and CDR region of the anti-PRLR antibodies obtained in this disclosure. Sequences prefixed with P were derived from phage display screening, sequences prefixed with H were derived from hybridoma screening, and sequences prefixed with HP were derived from phage libraries constructed from positive hybridoma clone cDNA.

[0140] [Table 2]

[0141] Example 6. Expression and purification of anti-PRLR antibody Primers were designed, and the VH and VL gene fragments of the antibodies shown in Table 2 were obtained by PCR amplification. Overlap PCR was performed with the constant region gene fragments CH1-Hinge-CH2-CH3 and CL of the human IgG1 heavy chain and light chain, respectively, to obtain fusion DNA sequences. These were inserted into the mammalian expression vector pCDNA3.1 to construct a plasmid vector expressing the heavy and light chains of the chimeric antibody. After sequence validation confirmed the correctness of the sequences, the plasmid vector was extracted using an endotoxin-removed plasmid extraction kit and stored at -20°C for use. Expi293F was stored in approximately 4x10⁻⁶ units. 6The plasmids expressing the light and heavy chains of the antibody, respectively, were diluted to a cell / mL density and co-transfected into Expi293F cells using PEI40000 (polysciences) transfection reagent. After 4 days, the supernatant of the cell culture medium was collected, centrifuged at high speed, and the supernatant was collected again and filtered through a 0.22 μm filter to remove any remaining cell debris. The filtered supernatant was purified using a Protein A column, the Protein A column was washed with PBS buffer to remove unwanted proteins, and the A280 value was lowered to stabilize at baseline. The target protein was then eluted with a 0.1 M sodium acetate solution at pH 3.2, the target protein peak was collected, and neutralized with a 1 M Tris-HCl solution at pH 8.0. After concentrating the sample, it was further purified using a gel chromatography column ENrich™ SEC650 (Bio-red), aggregates were removed, and individual peaks were collected. The collected samples were subjected to electrophoretic detection using 4-12% SDS-PAGE gradient gel, then packaged and stored at -80°C for use.

[0142] Example 7. Species-specific ELISA detection of anti-PRLR antibodies Recombinant PRLR extracellular domain antigen proteins from single mice, monkeys, and humans were diluted in PBS buffer (final concentration 1 μg / mL), and the antigen proteins were added to a 96-well high-adsorption plate (100 μL / well) and incubated overnight at 4°C. The following day, the 96-well plate (300 μL / well) was blocked with PBS blocking solution containing 3% skim milk powder and left at room temperature for 1 hour. The 96-well plate was then washed three times with PBS washing solution containing 0.1% Tween-20. 100 μL of purified target antibody (diluted 10 μg / mL in PBS) was added to each well and left at room temperature for 1 hour. The 96-well plate was washed three times, and 100 μL of 2000-fold diluted HRP-marked goat anti-mouse antibody (Thermo) was added to each well and incubated at room temperature for 1 hour. After washing the plate four times with washing solution, 50 μL of TMB chromogenic solution (Invitrogen) was added and allowed to develop for 2-5 minutes. The reaction was stopped by adding 1M sulfuric acid, and the absorbance at 450 nm was recorded using a microplate reader. Table 3 shows the species-specific ELISA test results for the antibodies obtained in this invention, with the control protein being a mesothelin recombinant protein with the same tag. As shown in Table 3, all obtained antibodies specifically bound to the human PRLR extracellular segment, and because the cynomolgus monkey PRLR extracellular segment and the human PRLR extracellular segment have a high degree of sequence agreement (agreement rate: 97.6%), differing by only five amino acid sites, most of the 23 antibody strains, except for antibodies 18, 21, and 23, also bound to the cynomolgus monkey PRLR extracellular segment. Furthermore, antibodies 3, 7, 9, 16, and 19 also bound to the mouse PRLR extracellular segment.

[0143] [Table 3]

[0144] Example 7. Affinity detection of anti-PRLR antibody The dynamic parameters (kon, koff, and KD) of antibody-PRLR ECD binding were analyzed using biofilm interferometry (OCTET R2, Sartorius). Protein A sensors (Octet® ProA Biosensors) were immersed in PBS buffer containing the analyte antibody (200 nM) along with 0.1% BSA, 0.05% Tween 20, pH 7.4 to bind the antibody to the sensor surface. After washing, the sensors were sequentially immersed in buffers containing individual PRLR antigens at different concentrations, and the affinity parameter KD was calculated using a 1:1 Langmuir model. As can be seen from Table 4, most antibodies were able to bind to the target antigen with high affinity, and the binding affinity ranged from nM to pM.

[0145] [Table 4]

[0146] Example 10. Humanization of anti-human PRLR monoclonal antibody Human antibody sequences with high homology to the target mouse antibody sequence were selected by sequence alignment and used as templates. The CDR region of the mouse antibody was determined using the IMGT numbering system, and the heavy chain and light chain CDR region sequences were transplanted into the human template, respectively. Simultaneously, the humanized sequences were obtained while retaining the following amino acids in their structural positions, such as Vernier Zone amino acids (Foote et al. J.Mol.Biol, 1992, 224, 487-499); amino acids located at the interface between the heavy chain and light chain that affect the pairing of the heavy chain and light chain in the structural model; and amino acids that affect the structure of the antibody CDR region (Vargas-Madrazo et al. J.Mol.Biol, 1995, 254, 497-504; AI-Lazikani et al. J.Mol.Biol, 1997, 273, 927-948) as mouse amino acids. 1. Humanization of clone P-PR-C05H Using common sequences of human antibodies as templates for the heavy and light chains, the mouse CDR region sequence was transplanted into the human template, while retaining the amino acid sites important for structure and function in the mouse amino acid sequence (Table 5), to obtain the P-PR-C05H-v1 heavy chain (SEQ ID NO: 20) and light chain sequence (SEQ ID NO: 40). The S57A mutation was applied to the heavy chain sequence of P-PR-C05H-v1 to obtain the humanized antibody P-PR-C05H-v2 (heavy chain sequence: SEQ ID NO: 21), and the antibody sequence was numbered according to the IMGT numbering rules.

[0147] [Table 5]

[0148] 2. Humanization of clone P-PR-C07H Using common sequences of human antibodies as templates for the heavy and light chains, the mouse CDR region sequence was transplanted into the human template, while retaining the amino acid sites important for structure and function in the mouse amino acid sequence (Table 6), thereby obtaining the P-PR-C07H-v1 heavy chain (SEQ ID NO: 22) and light chain sequence (SEQ ID NO: 40).

[0149] [Table 6]

[0150] 3. Humanization of clone HP-PR01D12 Using common sequences of human antibodies as templates for the heavy and light chains, the mouse CDR region sequence was transplanted into the human template, while retaining the amino acid sites important for structure and function in the mouse amino acid sequence (Table 7), thereby obtaining the HP-PR01D12-v heavy chain (SEQ ID NO: 23) and light chain sequence (SEQ ID NO: 41).

[0151] [Table 7]

[0152] Example 11. Affinity detection and epitope analysis of humanized PRLR-specific antibodies Single human or monkey PRLR ECD proteins were macbed with biotin, and the macbed proteins were diluted in PBS buffer (100 nM) at pH 7.4 containing 0.1% BSA and 0.05% Tween 20. Streptabidine sensors were immersed in the biotin-macbed protein solution to immobilize the target antigen, then washed with buffer. Subsequently, the sensors were sequentially immersed in buffers containing humanized antibodies at different concentrations (3.125–100 nM), bound for 2 minutes, and dissociated for 8 minutes. After subtracting the control sensor, dynamical curves were obtained for the binding of humanized antibodies, named P-PR-C05H-v2, P-PR-C07H-v, and HP-PR01D12-v, to human or monkey PRLR ECD. The affinity parameter KD was obtained by fitting to a 1:1 Langmuir model. As shown in Table 8, all three humanized antibodies can bind to human and monkey PRLR ECD proteins with high affinity.

[0153] [Table 8]

[0154] Using BLI, the antigen-binding epitopes of three humanized antibodies were analyzed. Biotin-tagged human mononucleotide PRLR ECDs were immobilized on streptavidin (SA) sensors. Anti-P-PR-C05H-v2 antibody, hP-PR-C07H-v antibody, and HP-PR01D12-v antibody were diluted to a final concentration of 100 nM using PBS buffer in 0.1% BSA, 0.05% Tween20, pH 7.4. The sensor was immersed in the first antibody sample until the signal saturated, and then immersed in the 100 nM second antibody. For first and second antibodies recognizing the same epitope, if the first antibody saturated the epitope on the PRLR surface, the second antibody could not bind to the PRLR, and the binding signal of the second antibody decreased in the presence of overlapping epitopes. For different epitopes, the binding signal of the second antibody was in close agreement with the reference (i.e., when the first antibody was in buffer). The epitope distribution was analyzed by the ratio of the binding signal of the second antibody to the reference. However, if the ratio <20%, the two antibodies have the same epitope; if 20% to 60%, there is partial overlap in the epitopes; and if it exceeds 60%, the epitopes do not completely overlap. The epitopes of the three humanized antibodies are shown in Table 9, and it was found that P-PR-C05H-v2 and HP-PR01D12-v have the same antigen-binding epitope, and P-PR-C07H-v is close to the antigen-binding epitope of P-PR-C05H-v2 and HP-PR01D12-v, but not a perfect match.

[0155] [Table 9]

[0156] Example 12. Receptor internalization assay mediated by a humanized PRLR-specific antibody. Immunofluorescence was used to detect the internalization of humanized antibodies P-PR-C05H-v2, P-PR-C07H-v, and HP-PR01D12-v after binding to PRLR. 10 μg / mL of humanized antibody was incubated with T47D cell lines (breast cancer cell lines endogenously expressing human PRLR) at 4°C for 1 hour. Excess antibody was washed away, and anti-human Fc antibody with a FITC fluorescent marker was added. The cells were incubated again at 4°C for another 1 hour. Excess antibody was washed away, fresh medium was added, and the control cells were placed at 4°C, while the internalization group cells were placed at physiological temperature (37°C, CO2 incubator). After 1 hour, the cells were removed, fixed with 4% paraformaldehyde, and the internalization of cell surface antigens was observed under a fluorescence microscope. As shown in Figure 1, internalized antibodies were clearly observed within the cells after only 1 hour of incubation at 37°C.

[0157] Example 13. Humanized PRLR-specific antibodies inhibit PRL-mediated PRLR activation and signal transduction. A luciferase reporter gene assay was used to measure whether humanized PRLR-specific antibodies inhibit prolactin (PRL)-mediated receptor dimer conformational changes and downstream signaling. Specifically, the plasmid pGL4.52 (Promega, #E465A), which encodes a STAT5-dependent luciferase reporter gene, was transfected into HEK293 cells that stably express PRLR using Lipofectamine LTX (Thermo, #15338-100). Twenty-four hours after transfection, the cells were digested with trypsin, the cell count was measured, and then 5 x 10⁶ cells were placed in a 96-well white plate. 4Cells were seeded at a ratio of individual cells / well, and a constant concentration of 5 nM PRL was mixed with various concentrations of humanized antibodies (50 pM to 100 nM) and added to the cells. The cells were incubated at 37°C for 5 hours. After incubation, luciferase activity in each well was measured using Bright-Glo® reagent (Promega, #E2610). The IC50 values ​​of humanized antibodies inhibiting PRL-mediated receptor activation were calculated. As shown in Table 10, P-PR-C05H-v2, P-PR-C07H-v, and HP-PR01D12-v each exhibit different abilities to inhibit PRL-mediated receptor activation, with P-PR-C07H-v completely inhibiting PRL-mediated receptor activation at a concentration of 100 nM (signal is baseline value). Furthermore, P-PR-C05H-v2, P-PR-C07H-v, and HP-PR01D12-v themselves did not activate PRLR at test concentrations (50 pM to 100 nM).

[0158] [Table 10]

[0159] Example 14. Detection of target cell killing mediated by anti-PRLR antibody-DT3C 293T cells and 293T-PRLR cells (5000 cells / 100 μL / well) were inoculated into 96-well cell culture plates. After 24 hours of incubation, anti-PRLR antibody and DT3C protein were added to fresh medium and incubated at 37°C. The final concentrations of anti-PRLR antibody were set to 0.1, 1, and 10 μg / mL, and the corresponding DT3C concentrations were set to 0.2, 2, and 20 μg / mL. After incubation, the medium was aspirated from the original 96-well plate, and the target samples were added to the cells. Simultaneously, a medium control group and a DT3C control group were established. The cells were cultured at 37°C for 24 hours. After culturing, 10 μL of CCK8 solution (purchased from TransGen Biotech, product number FC101-02) was added to each well. 96-well plates wrapped in aluminum foil were incubated in a 37°C incubator for 1 hour, and then the absorbance at 450 nm was measured using a Synergy H1 microplate reader (purchased from BioTek). As shown in Figure 2, all three humanized antibodies effectively mediated that DT3C killed PRLR-positive target cells but not cells that did not express PRLR.

[0160] [Table 11]

[0161] TIFF2026516781000012.tif241170

[0162] TIFF2026516781000013.tif148170

Claims

1. An antibody that targets PRLR or its antigen-binding fragment, or a variant that has at least 85% sequence identity with the antibody or its antigen-binding fragment while retaining its PRLR-binding activity, wherein the antibody targets an extracellular segment that targets the prolactin receptor; Preferably, the extracellular segment comprises amino acids at positions 1-210 of SEQ ID NO: 99 or 101; More preferably, the antibody comprises three HCDRs in the heavy chain variable region indicated by any one of SEQ ID NO: 1-23, and / or three LCDRs in the light chain variable region indicated by any one of SEQ ID NO: 24-41.

2. The HCDR1 of the antibody comprises one selected from: SEQ ID NO: 42-52, or a sequence having at least 85% sequence identity thereto; and / or The HCDR2 of the antibody comprises one selected from: SEQ ID NO: 53-66, or a sequence having at least 85% sequence identity thereto; and / or The HCDR3 of the antibody comprises one selected from: SEQ ID NO: 67-75, or a sequence having at least 85% sequence identity thereto; and / or The LCDR1 of the antibody comprises one selected from: SEQ ID NO: 76-83, or a sequence having at least 85% sequence identity thereto; and / or The LCDR2 of the antibody comprises one selected from: SEQ ID NO: 84-90, or a sequence having at least 85% sequence identity thereto; and / or The LCDR3 of the antibody comprises one selected from the following: SEQ ID NO: 91-98, or a sequence having at least 85% sequence identity thereto; Preferably, the antibody comprises HCDR1, HCDR2 and HCDR3 of the antibodies shown in any one row of Table 1, or a sequence having at least 85% sequence identity thereto; and / or, the antibody comprises LCDR1, LCDR2 and LCDR3 of the antibodies shown in any one row of Table 1, or a sequence having at least 85% sequence identity thereto; The aforementioned antibodies include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which are shown in any one row of Table 1. The antibody or its antigen-binding fragment as described in claim 1.

3. The light chain variable region of the antibody includes a mouse or human light chain FR region, and the heavy chain variable region of the antibody includes a mouse or human heavy chain FR region; Preferably, the heavy chain variable regions FR1, FR2, FR3, and FR4 of the antibody are each independently selected from the heavy chain variable regions FR1, FR2, FR3, and FR4 shown in any one of SEQ ID NO: 1-23, and / or the light chain variable regions FR1, FR2, FR3, and FR4 of the antibody are each independently selected from the light chain variable regions FR1, FR2, FR3, and FR4 shown in any one of SEQ ID NO: 24-41; More preferably, the antibody includes VH, VL, or VH and VL of the antibody shown in any one row of Table 1, or a sequence having at least 85% sequence identity thereto. An antibody or its antigen-binding fragment as described in 1 or 2.

4. The antibody further comprises a heavy chain constant region and / or a light chain constant region; and / or The antibody is a multispecific antibody; and / or The antibody is a monoclonal antibody; and / or The aforementioned antibody is either a chimeric antibody or a fully human antibody. An antibody or its antigen-binding fragment as described in 1 or 2.

5. A fusion protein or antibody complex comprising an antibody or antigen-binding fragment described in any one of claims 1-4.

6. (1) an antibody or antigen-binding fragment according to any one of claims 1-4, or a coding sequence of a fusion protein or antibody complex according to claim 5; (2) Complementary sequence of (1) A polynucleotide selected from among them.

7. A protein that expresses an antibody or antigen-binding fragment described in any one of claims 1-4, or is a fusion protein or antibody complex described in claim 5, or comprises a polynucleotide described in claim 6; Preferably, the nucleic acid construct is a vector. Nucleic acid construct.

8. A phage comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-4, or a library comprising such phage.

9. (1) Express and / or secrete an antibody or antigen-binding fragment described in any one of claims 1-4; (2) comprising the polynucleotide described in claim 6; and / or (3) comprising the nucleic acid construct described in claim 7 A host cell characterized by the following: Preferably, the host cell is a mammalian cell.

10. A method for producing an antibody or an antigen-binding fragment, comprising culturing host cells as described in claim 9 under conditions suitable for producing an antibody or an antigen-binding fragment thereof, and optionally purifying the antibody or an antigen-binding fragment thereof from the culture.

11. A pharmaceutical composition comprising an antibody or its antigen-binding fragment, fusion protein, antibody complex, polynucleotide, nucleic acid construct, phage or host cell, and a pharmaceutically acceptable auxiliary material, as described in any one of claims 1 to 4.

12. An antibody or antigen-binding fragment according to any one of claims 1-4, a fusion protein or antibody complex according to claim 5, a polynucleotide according to claim 6, a nucleic acid construct according to claim 7, or an application of a host cell according to claim 9 in the preparation of a pharmaceutical product for preventing or treating a disease; Preferably, the diseases include cancer, endometriosis, alopecia, osteoporosis, obesity, and benign lesions caused by PRLR activation; More preferably, the cancer is a cancer associated with PRLR; More preferably, the cancer is selected from ovarian cancer, melanoma, prostate cancer, colorectal cancer, stomach cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain tumor, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies.

13. A kit for detecting PRLR used for evaluating the therapeutic effect of a drug or diagnosing cancer, comprising an antibody or antigen-binding fragment according to any one of claims 1-4, a fusion protein or antibody complex according to claim 5, a polynucleotide according to claim 6, a nucleic acid construct according to claim 7, a phage according to claim 8, or a host cell according to claim 9; Preferably, the kit further includes reagents for detecting the binding of PRLR to an antibody or its antigen-binding fragment, fusion protein, or antibody complex.

14. Application of an antibody or antigen-binding fragment according to any one of claims 1-4, or a fusion protein or antibody complex according to claim 5, a polynucleotide according to claim 6, a nucleic acid construct according to claim 7, a phage according to claim 8, or a host cell according to claim 9, in the detection of PRLR in a sample, evaluation of the therapeutic effect of a drug, or in the manufacture of a kit used in a cancer diagnostic kit.

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