Prolactin receptor antibodies and uses thereof

Abstract

The invention relates to a prolactin receptor antibody and application thereof, in particular to a PRLR targeting antibody or an antigen binding fragment thereof, or a variant which has at least 85% sequence identity with the antibody or the antigen binding fragment thereof and retains PRLR binding activity thereof, and the antibody targets an extracellular fragment of a prolactin receptor. The antibodies of the invention bind to PRLR with high affinity.

Description

Technical Field

[0001] This invention relates to the fields of biology and medicine; more specifically, this invention relates to an antibody that specifically binds to the prolactin receptor and its uses. Background Technology

[0002] The prolactin receptor (PRLR) is a single-transmembrane protein, a type I cytokine receptor, and is homologous to interleukins 2, 3, 4, 6, and 7, erythropoietin, and granulocyte-macrophage colony-stimulating factor receptors. The natural ligand of the prolactin receptor, prolactin, is a polypeptide hormone composed of 199 amino acids, primarily secreted by lactocytes in the human pituitary gland. Upon binding to the homodimeric form of the prolactin receptor, prolactin induces a conformational change in the receptor dimer, thereby activating Jak2 bound to the intracellular segment of the receptor. Jak2 catalyzes the formation of an active phosphorylated dimer of Stat5, which then enters the nucleus to regulate downstream gene expression. Besides the main Jak2-Stat5 signaling pathway, prolactin receptor activation can also activate Src, promoting cell proliferation, and activate MARK, among other things. Furthermore, growth factor hormones and placental prolactin can also bind to and activate the prolactin receptor. Therefore, PRLR activation can mediate a variety of biological functions and is a potential target for disease intervention in some benign or malignant diseases.

[0003] Prolactin receptors mediate normal physiological functions, including promoting cell growth, differentiation, development, and lactation. Human prolactin receptor cDNA was initially isolated from liver and breast cancer libraries. The mature protein has a full length of 598 amino acids. Due to different mRNA trimming, prolactin receptors exist in different subtypes: L, I, S1a, and S1b. These subtypes share the same extracellular segment and different lengths of intracellular segments. Prolactin receptors are considered potential therapeutic targets for breast and prostate cancer, but their role in breast cancer development and progression remains controversial. Conversely, extrapituitary prolactin (PRL), synthesized outside the pituitary gland through autocrine and / or paracrine processes, is thought to be involved in prostate tumorigenesis. In mouse models, prostate overexpression of PRL can lead to benign prostatic hyperplasia, intraepithelial neoplasia, and even prostate adenoma. Epidemiologically, the expression of PRL and phosphorylated Stat5 in the prostate tissue of patients is positively correlated with tumor malignancy and disease invasion.

[0004] Prolactin receptors (PRLRs) are not only potential targets for malignant tumors but also important targets in various benign lesions. Clinical studies have shown that enhanced PRLR-mediated signaling is associated with endometriosis in women. An exploratory clinical study showed that treatment with the dopamine agonist quincolitide (a prolactin synthesis regulator) for more than 4 months significantly reduced the size of peritoneal endometriotic lesions in women diagnosed with endometriosis and hyperprolactinemia. Furthermore, prolactin can interfere with the hair follicle cycle, and elevated prolactin levels in the blood are associated with hair loss. Expression of the prolactin receptor in hair follicles has been demonstrated, and prolactin can induce the hair follicle cycle into the anagen phase. In mouse models, dopamine receptor agonists (PRL inhibitors) can promote hair growth. In conclusion, PRLRs are promising drug targets, and antibodies or antigen-binding fragments targeting PRLRs can be used for various PRLR-related diseases, showing broad market potential. Summary of the Invention

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

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

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

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

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

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

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

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

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

[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:

[0015] (1) SEQ ID NO: 43, SEQ ID NO: 55, and SEQ ID NO: 67,

[0016] (2) SEQ ID NO: 43, SEQ ID NO: 66, and SEQ ID NO: 67,

[0017] (3) SEQ ID NO: 44, SEQ ID NO: 56 and SEQ ID NO: 68,

[0018] (4) SEQ ID NO: 50, SEQ ID NO: 63 and SEQ ID NO: 73, and / or

[0019] The LCDR1, LCDR2, and LCDR3 of the antibody are selected from any one of the following groups or sequences having at least 85% sequence identity with them:

[0020] (1) SEQ ID NO: 77, SEQ ID NO: 84 and SEQ ID NO: 91,

[0021] (2) SEQ ID NO:82, SEQ ID NO:90 and SEQ ID NO:97.

[0022] In one or more embodiments, the antibody contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antibodies shown in any row of Table 1.

[0023] In one or more embodiments, the light chain variable region of the antibody comprises a mouse or human light chain FR region. In one or more embodiments, the heavy chain variable region of the antibody comprises a mouse or human heavy chain FR region.

[0024] 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 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 of SEQ ID NO:24-41.

[0025] In one or more embodiments, the FR region of the heavy chain variable region of the antibody is selected from the FR region of the heavy chain variable region shown in any of SEQ ID NO: 3, 5, 16, 20-23.

[0026] In one or more embodiments, the FR region of the light chain variable region of the antibody is selected from the FR region of the light chain variable region shown in any of SEQ ID NO: 26, 28, 36, 40, 41.

[0027] In one or more embodiments, the antibody contains the VH, VL, or VH and VL of the antibody shown in any row of Table 1, or a sequence having at least 85% sequence identity with it.

[0028] In one or more embodiments, the VH of the antibody has a sequence shown in any of SEQ ID NO: 3, 5, 16, 20-23 or a sequence having at least 85% sequence identity with it. Alternatively, or furthermore, the VL of the antibody has a sequence shown in any of SEQ ID NO: 26, 28, 36, 40, 41 or a sequence having at least 85% sequence identity with it.

[0029] In one or more embodiments, the antibody includes:

[0030] VH is shown in SEQ ID NO:3, and VL is shown in SEQ ID NO:26.

[0031] VH is shown in SEQ ID NO:5, and VL is shown in SEQ ID NO:28.

[0032] VH is shown in SEQ ID NO:16, and VL is shown in SEQ ID NO:36.

[0033] VH is shown as SEQ ID NO:20 or 21, and VL is shown as SEQ ID NO:40.

[0034] VH is shown as SEQ ID NO:22, VL is shown as SEQ ID NO:40, or

[0035] VH is shown as SEQ ID NO:23, and VL is shown as SEQ ID NO:41.

[0036] In one or more embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0037] In one or more embodiments, the antibody is a multispecific antibody, preferably a bispecific antibody.

[0038] In one or more embodiments, the antibody is a monoclonal antibody.

[0039] In one or more embodiments, the antibody is a chimeric antibody or a fully human antibody.

[0040] The present invention also provides a fusion protein or antibody conjugate comprising the antibody or its antigen-binding fragment described herein.

[0041] This invention also provides polynucleotides selected from:

[0042] (1) The coding sequence of the antibody or its antigen-binding fragment, fusion protein, or antibody conjugate described in any of the embodiments herein;

[0043] (2)(1) complementary sequences.

[0044] The present invention also provides a nucleic acid construct that expresses an antibody or its antigen-binding fragment, fusion protein, or antibody conjugate as described in any embodiment herein, or contains a polynucleotide as described in any embodiment herein.

[0045] In one or more embodiments, the nucleic acid construct is a vector, such as an integration vector, a cloning vector, or an expression vector.

[0046] The present invention also provides a phage containing an antibody or an antigen-binding fragment thereof as described in any embodiment herein, or a library containing such a phage.

[0047] In one or more embodiments, the antibody or its antigen-binding fragment is presented on the surface of the phage.

[0048] The present invention also provides a host cell, wherein:

[0049] (1) Expressing and / or secreting the antibody or its antigen-binding fragment as described in any of the embodiments herein;

[0050] (2) Contains the polynucleotides described herein; and / or

[0051] (3) Includes the nucleic acid constructs described in this article.

[0052] In one or more embodiments, the host cell is selected from prokaryotic cells or eukaryotic cells.

[0053] In one or more embodiments, the host cell is a mammalian cell.

[0054] The present invention also provides a method for generating an antibody or an antigen-binding fragment thereof, comprising: culturing the host cells described herein under conditions suitable for generating the antibody or an antigen-binding fragment thereof, and optionally purifying the antibody or the antigen-binding fragment thereof from the culture.

[0055] The present invention also provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof described herein, a fusion protein, an antibody-drug conjugate, a polynucleotide, a nucleic acid construct, a bacteriophage or a host cell, and pharmaceutically acceptable excipients.

[0056] In one or more embodiments, the excipient is a carrier, diluent, or excipient.

[0057] In one or more embodiments, the pharmaceutical composition is used to treat cancer.

[0058] In one or more embodiments, the cancer is a PRLR-related cancer. Preferably, the cancer is selected from ovarian cancer, melanoma, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, thyroid cancer, and hematologic malignancies. Hematologic malignancies are selected from myeloma, chronic leukemia, and acute leukemia.

[0059] The present invention also provides the use of the antibody or antigen-binding fragment thereof, fusion protein, antibody conjugate, polynucleotide, nucleic acid construct or host cell described in any embodiment herein in the preparation of a medicament for the prevention or treatment of a disease.

[0060] In one or more embodiments, the disease includes: cancer, endometriosis, hair loss, osteoporosis, obesity, and benign lesions caused by PRLR activation.

[0061] In one or more embodiments, the cancer is a PRLR-related cancer. Preferably, the cancer is selected from ovarian cancer, melanoma, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, thyroid cancer, and hematologic malignancies. Hematologic malignancies are selected from myeloma, chronic leukemia, and acute leukemia.

[0062] The present invention also provides a method for inhibiting the growth of tumor cells in a subject, inhibiting benign lesions caused by PRL activation of PRLR, or treating or preventing disease, the method comprising administering to a patient in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof, fusion protein, antibody conjugate or pharmaceutical composition as described in any embodiment of the present invention.

[0063] In one or more embodiments, the disease includes: cancer, endometriosis, hair loss, osteoporosis, obesity, and benign lesions caused by PRLR activation.

[0064] In one or more embodiments, the cancer is a PRLR-related cancer. Preferably, the cancer is selected from ovarian cancer, melanoma, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, thyroid cancer, and hematologic malignancies. Hematologic malignancies are selected from myeloma, chronic leukemia, and acute leukemia.

[0065] The present invention also provides a kit for detecting PRLR, used to evaluate the efficacy of drug treatment or diagnose cancer, the kit comprising an antibody or its antigen-binding fragment as described in any embodiment herein, a fusion protein, an antibody-drug conjugate, a polynucleotide, a nucleic acid construct, a bacteriophage, or a host cell.

[0066] In one or more embodiments, the kit further includes reagents for detecting the binding of PRLR to an antibody or its antigen-binding fragment, fusion protein, or antibody-drug conjugate. For example, reagents for detecting the binding by an enzyme-linked immunosorbent assay (ELISA).

[0067] In one or more embodiments, the detection binding reagent is a detectable marker, such as biotin, that can be linked to an antibody or its antigen-binding fragment, fusion protein, or antibody-drug conjugate. The detectable marker is either linked to the antibody or its antigen-binding fragment or is present separately in the kit.

[0068] This invention also provides a non-diagnostic method for detecting the presence of PRLR in a sample. The method includes: incubating the sample with an antibody or its antigen-binding fragment, fusion protein, or antibody-drug conjugate as described in any embodiment herein; and detecting the binding of PRLR to the antibody or its antigen-binding fragment, fusion protein, or antibody-drug conjugate, thereby determining the presence of PRLR in the sample. The detection is performed using an enzyme-linked immunosorbent assay (ELISA).

[0069] The present invention also provides the use of the antibody or its antigen-binding fragment, fusion protein, or antibody-drug conjugate described in any embodiment herein in the preparation of a kit for detecting PRLR in a sample, evaluating the efficacy of drug treatment, or diagnosing cancer. Attached Figure Description

[0070] Figure 1 Immunofluorescence detection of PRLR endocytosis mediated by humanized PRLR antibodies disclosed in this paper.

[0071] Figure 2 This study aims to detect the killing of PRLR-positive cell lines mediated by the humanized PRLR antibody disclosed in this paper. Detailed Implementation

[0072] Unless otherwise defined, the present invention will be practiced using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of the art. These techniques are well explained in the literature, 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 R.R. Freshney, 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., 1987 edition and its periodically updated editions); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0073] The prolactin receptor (PRLR) is a single-transmembrane protein, a type I cytokine receptor, and shares homologs with interleukin-2, 3, 4, 6, and 7, erythropoietin, and granulocyte-macrophage colony-stimulating factor receptors. The prolactin receptor binds to human prolactin, growth factor hormones, and placental prolactin, mediating various biological functions, including cell proliferation, differentiation, and lactation. Abnormal activation of the prolactin receptor is associated with several human diseases, such as tumors, endometriosis, hair loss, hyperprolactinemia, and osteoporosis.

[0074] This invention utilizes the extracellular domain of the PRLR protein as an epitope peptide to obtain anti-human PRLR hybridoma monoclonal antibodies. Then, phage display technology is used to screen antibody gene libraries and perform humanization modifications, thereby obtaining a series of PRLR-specific antibodies. Antibodies with high affinity, high specificity, and high functional activity are then identified using ELISA, affinity assays, receptor internalization tests, blocking assays, and target cell killing assays. The antibodies or their antigen-binding fragments exhibit good safety and targeting properties, and can specifically bind to the extracellular domain of the PRLR protein.

[0075] Antibody

[0076] In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, and Fv. In this document, "antibody" and "immunoglobulin" are used interchangeably.

[0077] Traditional "antibodies" contain a basic four-chain antibody unit, a heterotetrameric glycoprotein composed 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 (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for μ and ε isoforms) constant domains (CH), and a hinge region located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. Pairs of VH and VL together form an antigen-binding site. For information on the structure and properties of different classes of antibodies, see 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 can be classified into one of two distinct types, called κ and λ, based on their constant domain amino acid sequences. Based on relatively minor differences in CH sequences and functions, the γ and α types can be further subdivided into subclasses, for example, those expressed in humans: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2. The antibody heavy chain may further include a heavy chain constant region comprising the constant regions of human or murine IgG1, 2, 3, and 4, or mutant sequences thereof. The antibody light chain may further include a light chain constant region comprising the constant regions of human or murine κ and λ chains, or mutant sequences thereof.

[0078] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0079] The term "variable" refers to the wide variation in certain segments within a variable domain within the antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire amino acid range spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (present in both light and heavy chain variable domains): HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain, and LCDR1, LCDR2, and LCDR3 in the light chain variable domain. More highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a loop and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FR regions and, together with the HVRs of the other chain, contribute 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. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity. Several variable region annotation schemes exist for antibodies, including Chothia, Kabat, IMGT, and Contact. This article uses the IMGT annotation scheme as an example.

[0080] The “Fc region” (crystallizable fragment region), “Fc domain”, or simply “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of the two antibody heavy chains; the Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2–4) in each polypeptide chain. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where the numbering is based on the EU index, as in Kabat. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

[0081] An "antigen-binding fragment" typically refers to a portion of an antibody molecule containing amino acids responsible for the specific binding between the antibody and the antigen. The part of the antigen that the antibody specifically recognizes and binds to is called an "epitope," as described above. As mentioned above, the antigen-binding domain typically includes a variable region (VL) and a variable region (VH) of the antibody light chain; however, it does not necessarily have to include both. Examples of antibody fragments include Fab, Fab', F(ab'), F(ab')2, Fd, and Fv fragments; disulfide-linked Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment whose half-life should be increased through chemical modification or incorporation into liposomes. Fd fragments, for example, have two VH regions and typically retain some antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light chain (CL) and CH1 domains; (2) F(ab')2 fragments, bivalent fragments having two Fab fragments connected by disulfide bridges of hinge regions; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains of antibody single arms; (5) dAb fragments (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,” Nature 341:544-546 (1989), which are incorporated herein by reference in their entirety), having a VH domain; (6) separate complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv), for example derived from scFV-libraries. Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be conjugated using a recombination method via synthetic linkers. These linkers allow for the preparation of a single protein chain (called a single-chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (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 their function is evaluated in the same manner as that of intact antibodies.

[0082] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently linked heavy chain variable domain and a light chain variable domain. Six hypervariable rings (three from the heavy chain and three from the light chain) protrude from the folds of these two domains, contributing the amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than a complete binding site. A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing antibody VH and VL domains linked together into a single polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired antigen-binding structure. For heavy chain antibodies or nanobodies, scFv is VHH.

[0083] In this document, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Compared to polyclonal antibody formulations (which typically consist of different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized through hybridoma culture, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the invention can be generated by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for generating human or human-like antibodies from animals having partial or whole human immunoglobulin loci or genes encoding human immunoglobulin sequences, single-cell sequencing methods.

[0084] Monoclonal antibodies also include “chimeric” antibodies in this article, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity.

[0085] The term "mouse antibody" in this disclosure refers to a mouse-derived monoclonal antibody that binds to human PRLR, prepared in accordance with the knowledge and skills of the art. Preparation involves injecting the test subject with the PRLR antigen, followed by isolating and expressing the antibody with the desired sequence or functional characteristics. In a preferred embodiment of this disclosure, the mouse PRLR antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a mutant sequence thereof, or further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3 or a mutant sequence thereof.

[0086] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. To establish a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first created. Then, the variable region gene is cloned from mouse hybridoma cells, and the constant region gene of the human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a vector. Finally, the chimeric antibody molecule is expressed in eukaryotic or prokaryotic cells. In a preferred embodiment of the invention, the antibody light chain of the chimeric antibody further includes the 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 the heavy chain constant region of a human IgG1, IgG2, IgG3, or IgG4, or a mutant sequence thereof, preferably including the human IgG1 heavy chain constant region.

[0087] The “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains at least a sequence derived from a non-human immunoglobulin. Therefore, a “humanized antibody” generally refers to a non-human antibody with a variable domain framework region that exchanges sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody (except the CDR) is encoded by human-derived polynucleotides or is identical to that antibody (except the CDR). The CDR (some or all of which are encoded by nucleic acids derived from non-human organisms) is grafted into the β-sheet backbone of the human antibody's variable region to produce an antibody whose specificity is determined by the grafted CDR. The term “humanized antibody” is also known as a CDR grafted antibody. Methods for producing this type of antibody are well known in the art, such as using mice with genetically engineered immune systems. Human germline antibody variable region framework (FR) sequences are available from the ImMunoGeneTics (IMGT) website at http: / / imgt.cines.fr. The common sequence of human 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 antibody activity along with a decrease in immunogenicity, the variable region of the human antibody can be reverse-mutated (reverted to a mutation) to maintain activity. In this invention, the antibody includes a humanized variant.

[0088] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies generated by humans and / or produced using any of the techniques disclosed herein for generating human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

[0089] The term “binding with PRLR” in this disclosure refers to the ability to interact with human PRLR.

[0090] The term "antigen-binding epitope" in this disclosure refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment of this disclosure.

[0091] In some embodiments, the present invention also provides nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that compete with the antigen-binding region of any antibody of the present invention for binding to the same epitope on the PRLR, i.e., nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that can cross-compete with the antigen-binding region of any antibody of the present invention for binding to the PRLR.

[0092] In a specific embodiment of the present invention, the anti-PRLR antibody contains any of the CDR groups shown in any row of Table 1:

[0093] Table 1

[0094]

[0095] The FR1, FR2, FR3, and FR4 of the antibody described herein can be independently selected from the FR1, FR2, FR3, and FR4 of the VH or VL 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 of SEQ ID NO:1-23; and 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 of SEQ ID NO:24-41.

[0096] In one or more embodiments, the antibody contains VH, VL, or VH and VL of the antibody shown in any row of Table 1. Preferably, VH is as shown in SEQ ID NO:3 and VL is as shown in SEQ ID NO:26, or VH is as shown in SEQ ID NO:5 and VL is as shown in SEQ ID NO:28, or VH is as shown in SEQ ID NO:16 and VL is as shown in SEQ ID NO:36, or VH is as shown in SEQ ID NO:20 or 21 and VL is as shown in SEQ ID NO:40, or VH is as shown in SEQ ID NO:22 and VL is as shown in SEQ ID NO:40, or VH is as shown in SEQ ID NO:23 and VL is as shown in SEQ ID NO:41.

[0097] 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 a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a sequence having at least 85% sequence identity with it; the light chain of the antibody comprises a light chain constant region of human κ or λ chains, or a sequence having at least 85% sequence identity with it. Preferably, the heavy chain of the antibody comprises a heavy chain constant region of human IgG1, or a sequence having at least 85% sequence identity with it, and the light chain comprises a light chain constant region of human κ chains, or a sequence having at least 85% sequence identity with it.

[0098] In this article, the antibody can be a multispecific antibody comprising one, two, or more chains or antigen-binding fragments of the antibody described herein. Multispecificity can be against PRLR and another antigen, or against two different epitopes of PRLR.

[0099] This invention also includes derivatives and analogs of the antibodies described herein. “Derivatives” and “analytes” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The derivatives or analogs of this invention may be (i) polypeptides having substituents in one or more amino acid residues, or (ii) polypeptides formed by fusing a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these derivatives and analogs are within the scope well known to those skilled in the art.

[0100] The term "mutation" in the "mutated 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 replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, in general, a single amino acid substitution 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, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0101] Without substantially affecting antibody activity, those skilled in the art can modify the antibody sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter protein function. For example, substitutions of amino acids with similar properties in the FR and / or Fc regions. 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. For example, adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. These are all considered to be included within the scope of protection of this invention.

[0102] The variants of the antibodies described herein include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention. In some embodiments, the sequences of the variants described herein may have at least 95%, 96%, 97%, 98%, or 99% homology with their source sequences. The sequence homology described herein can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology with the CDRs identified herein.

[0103] The antibodies of the present invention can be prepared using methods conventional in the art, such as hybridoma technology and phage display technology. Alternatively, the antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with the sequence encoding the antibody of the present invention, and then the host cells can be cultured and the antibody purified. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., HepG2).

[0104] This disclosure also provides a fusion protein comprising the PRLR extracellular region and a fragment of the mouse antibody IgG2a Fc. A fusion protein is a protein product obtained by co-expressing two genes through DNA recombination. The recombinant PRLR extracellular region Fc fusion protein is a fusion protein that co-expresses the PRLR extracellular region and a fragment of the mouse antibody IgG2a Fc through DNA recombination. The PRLR extracellular region refers to the portion of the PRLR protein expressed outside the cell membrane.

[0105] This invention also includes fusion proteins containing the anti-PRLR antibody described herein and other peptides. In some embodiments, the other peptides are located at the N-terminus and / or C-terminus of the antibody. In some embodiments, the other peptides include peptides that localize the antibody to different organelles, tags for purification or for immune responses, transmembrane proteins or their transmembrane regions, chimeric antigen receptors or components thereof (extracellular domains, hinge regions, transmembrane regions, signal transduction domains, co-stimulatory domains, etc.).

[0106] This invention also includes antibody-drug conjugates containing the anti-PRLR antibody described herein. These conjugates are drugs in which a biologically active cytotoxic drug is chemically linked to an antibody, and the antibody acts as a carrier to target and deliver the cytotoxic drug to the target cell to exert its effect. Those skilled in the art can select the drug conjugated with the anti-PRLR antibody described herein according to their needs and circumstances.

[0107] Nucleic Acids

[0108] This invention also provides polynucleotides encoding the antibodies described herein. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. This invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but different nucleotide sequences. RNA can be mRNA expressing antibodies in vivo and / or in vitro.

[0109] Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0110] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, a long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein. The sequences of the various parts of the fusion protein can be obtained as described above and then ligated to obtain the full-length fusion protein.

[0111] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms. Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis. The portions of the fusion protein can be sequentially cloned into a vector or integrated into a full-length fusion protein before cloning.

[0112] This invention also relates to nucleic acid constructs containing the polynucleotide sequences described herein, and one or more regulatory sequences operatively linked to these sequences, such as regulatory sequences suitable for expressing DNA or RNA as antibodies in vivo or in vitro. The polynucleotide sequences described herein can be manipulated in various ways to ensure the expression of the antibodies. The nucleic acid constructs can be manipulated prior to insertion into a vector, depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0113] The regulatory sequence can be a suitable promoter sequence. Promoter sequences are typically operatively linked to the coding sequence of the protein to be expressed. A promoter can be any nucleotide sequence that exhibits transcriptional activity in the chosen host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that host cell. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the use of inducible promoters may also be considered. The use of inducible promoters provides a molecular switch that can turn on the expression of the polynucleotide sequence operatively linked to the inducible promoter during time-limited expression and turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0114] The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, the untranslated region of mRNA important for translation by the host cell. The leader sequence is operatively linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention.

[0115] Other regulatory sequences suitable for expressing DNA or RNA in vivo or in vitro are common knowledge in this field.

[0116] In some embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector. Expression of the polynucleotide sequence of the present invention is typically achieved by operably ligating the polynucleotide sequence of the present invention to an expression vector. A typical cloning vector contains transcription and translation terminators, a start sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence. An integration vector contains components for integrating the target sequence into the cellular genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These 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 containing donor and acceptor splicing sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-connector region for inserting a nucleic acid encoding an antibody to be expressed, and optional marker elements. An example of an expression vector is pcDNA3.1.

[0117] Furthermore, the type of vector is not limited; for example, plasmids, phage particles, phage derivatives, animal viruses, and entrapments can be modified depending on the host cell to be introduced. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0118] To assess the expression of a polypeptide or a portion thereof, the vector introduced into the cell may also contain one or both of an optional marker gene or reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector.

[0119] cell

[0120] The host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: *Escherichia coli*, *Streptomyces* spp.; bacterial cells of *Salmonella typhimurium*; fungal cells such as yeast; insect cells of *Drosophila* S2 or Sf9; animal cells of CHO, COS7, 293, etc. Examples of mammalian cells include immune cells, preferably immune effector cells. "Immune effector cells" are immune cells capable of performing immune effector functions, including: T cells, NK cells, peripheral blood mononuclear cells (PBMCs), neutrophils, eosinophils, and hematopoietic stem cells. The T cells suitable for this invention can be of various types from various sources.

[0121] Methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be transferred into cells by physical, chemical, or biological methods. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used. In some embodiments, transduced or transfected immune effector cells proliferate in vitro after the introduction of nucleic acids or vectors.

[0122] The obtained transformants can be cultured using conventional methods to express the antibody encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0123] The peptides used in the above methods can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, recombinant proteins can be separated and purified using various separation methods based on 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, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. For example, column chromatography using a Protein A or G Sepharose FF column can be used to wash away non-specifically bound components, followed by elution of bound antibodies with acidic buffer, detection of antibody fragments using SDS-PAGE, and the collected antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as gel filtration chromatography or ion exchange. The resulting product should be immediately frozen, such as at 80°C, or lyophilized.

[0124] Uses and methods

[0125] By constructing a nanobody library, the inventors screened nanobodies and their variants that could bind to PRLR. The binding ability of these antibodies to the antigen was verified through protein-level binding assays, affinity assays, competitive blocking experiments, and tissue cross-reactivity studies.

[0126] All aspects of the antibodies, coding sequences, nucleic acid constructs, and cells described herein can be used to prepare drugs for the prevention or treatment of the various conditions and diseases described herein, which are diseases or conditions related to PRLR expression, referring to diseases directly or indirectly caused by abnormal PRLR expression, usually referring to diseases caused by PRLR overexpression, such as cancer, including but not limited to: acute lymphoblastic leukemia (ALL), chronic lymphoblastic 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); relapsed or refractory follicular lymphoma (r / r FL), etc.

[0127] This article also provides a pharmaceutical composition comprising any one or more of an antibody or its antigen-binding fragment, a fusion protein, a nucleic acid molecule, a nucleic acid construct, and a cell, and pharmaceutically acceptable excipients.

[0128] The antibodies, nucleic acids, or cells of the present invention can be administered alone or as pharmaceutical compositions in combination with diluents and / or other components such as associated cytokines or cell populations. In this regard, pharmaceutical compositions can be prepared by mixing an active pharmaceutical agent of desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dose and concentration used and may include at least one of 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), and surfactants. Furthermore, in order for pharmaceutical compositions to be usable for in vivo administration, they must be sterile. Sterility of the pharmaceutical compositions can be achieved by filtration through a sterile filter membrane.

[0129] In some embodiments, the pharmaceutical composition may contain at least one additive selected from: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active pharmaceutical agent required for the specific indication to be treated. The specific amount of the additive may be adjusted as needed.

[0130] The pharmaceutical compositions of the present invention can be administered in amounts described as “immunologically effective,” “antitumor effective,” “tumor-suppressive effective,” or “therapeutic.” “Therapeutic” refers to a subject receiving the treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell invasion into surrounding organs, or a decrease in the rate of tumor metastasis or growth). When “immunologically effective,” “antitumor effective,” “tumor-suppressive effective,” or “therapeutic” is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient’s (subject’s) age, weight, tumor size, degree of infection or metastasis, and disease. Typically, pharmaceutical compositions comprising T cells described herein can be administered at 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 at these doses. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a specific patient can be easily determined by medical professionals by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0131] The composition can be administered in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The composition described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. The composition can also be injected directly into the tumor, lymph node, or site of infection.

[0132] In some embodiments of the invention, the compositions of the invention can be combined with other therapies known in the art. These therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, they can be combined with radiotherapy or chemotherapy agents known in the art for treating PRLR-mediated diseases.

[0133] In this article, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various cancer-related physiological symptoms.

[0134] The terms "patient," "subject," and "individual" are used interchangeably in this article to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and their transgenic species.

[0135] The present invention is described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the invention should not be construed as limited to the following examples, but should be interpreted as including any and all variations that become apparent from the teachings provided herein. The methods and reagents used in the examples, unless otherwise stated, are conventional methods and reagents in the art.

[0136] Diagnostics, tests and kits

[0137] The antibody of this invention, due to its high affinity for PRLR, can be used for assays, such as conjugation assays, to detect and / or quantify PRLR expressed in tissues or cells. The antibody can be used in studies further investigating the role of PRLR in disease. The method for detecting PRLR generally involves: obtaining cell and / or tissue samples; detecting the level of PRLR in the samples.

[0138] The PRLR antibody of this invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions related to PRLR. This invention provides methods for detecting the presence of PRLR in samples using classical immunohistochemical methods known to those skilled in the art. PRLR detection can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of PRLR include ELISA, FACS, RIA, etc.

[0139] For diagnostic applications, antibodies are typically labeled with detectable labeling groups. Suitable labeling 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, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites for secondary antibodies, metal-binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used in carrying out this invention.

[0140] Another aspect of the invention provides a method for detecting the presence of a test molecule that competes with the antibody of the invention for binding to PRLR. An example of such an assay would involve 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) would indicate that the test molecule is able to compete with the antibody for binding to PRLR. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of the antibody.

[0141] This invention also provides a detection kit for detecting PRLR levels, comprising a PRLR antibody, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0142] The present invention is described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the invention should not be construed as limited to the following examples, but should be interpreted as including any and all variations that become apparent from the teachings provided herein. The methods and reagents used in the examples, unless otherwise stated, are conventional methods and reagents in the art.

[0143] Example

[0144] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under the experimental conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

[0145] Example 1. Preparation of PRLR antigen and detection protein

[0146] Using the extracellular domain (amino acid sequences 25-234) of human PRLR protein (Uniprot: P16471) or the extracellular domain (amino acid sequences 25-234) of cynomolgus monkey PRLR protein (Uniprot: G8F5S4) as templates, antigens and detection proteins with different fusion tags were designed, including the avi-(his)6 tag (amino acids 211-231 of SEQ ID NO: 99) and the mouse IgG2a Fc (mFc) tag (amino acids 214-446 of SEQ ID NO: 100). The extracellular domain of human PRLR protein with the avi-(his)6 tag (hPRLR-ECD-avi-his) is shown in SEQ ID NO: 99. The extracellular domain of human PRLR protein with the mouse IgG2a Fc (mFc) tag (hPRLR-ECD-mFc) is shown in SEQ ID NO: 100. The extracellular domain of the PRLR protein of the cynomolgus monkey tagged with avi-(his)6 (cynoPRLR-ECD-avi-his) is shown in SEQ ID NO: 101.

[0147] A DNA sequence encoding the aforementioned protein (Shanghai Sangon Biotech) was synthesized and inserted into the pcDNA3.1 expression vector to construct a plasmid for expressing the recombinant protein. The target plasmid was transfected into Expi293F (Thermo) cells using PEI (Polyscience) to express the protein. Four days later, the cell culture supernatant was collected, centrifuged at high speed, and filtered through a 0.22 μm filter to remove residual cell debris.

[0148] The antigen protein was purified using either a nickel column or a Protein A column (GE) depending on the tag it carried. After purification, the sample was concentrated, identified by SDS-PAGE and mass spectrometry, and then aliquoted and stored at -80°C for later use.

[0149] Example 2. Construction of PRLR-expressing cell lines

[0150] DNA encoding the complete human PRLR sequence (Uniprot: P16471, 1-622) and carrying the P2A-GFP tag sequence (SEQ ID NO: 102, positions 625-882) was synthesized and inserted into the pcDNA3.1 expression vector. The purified plasmid was digested with Pvu I (NEB) and transfected into HEK293, Cos7, or CHO K1 cells using Lipofectamine LTX (Thermo). Twenty-four hours after transfection, 700-1000 μg / mL of G418 was added for selection. After continuous culture in G418-containing medium for two weeks, GFP expression was detected by flow cytometry, and GFP-positive cells were sorted. Stable PRLR-expressing cell lines HEK293-PRLR, CHOK1-PRLR, and Cos7-PRLR were constructed through single-cell sorting and amplification.

[0151] Example 3. Obtaining and preparing anti-human PRLR hybridoma monoclonal antibody

[0152] 1. Mouse immunization

[0153] Select 6-8 week old female Balb / c mice (Guangdong Yaokang Biotechnology Co., Ltd.), and mix the recombinant expressed protein antigen hPRLR-ECD-avi-his or hPRLR-ECD-mFc (protein concentration: 1-2 mg / mL) with an equal volume of adjuvant [options include: Freund's complete adjuvant (Sigma, F5881-10 mL), Freund's incomplete adjuvant (344291-10 mL), TiterMax Gold adjuvant (Sigma, T2684)]. [Alum (Thermo, 77161, etc.)] were thoroughly mixed for immunization. For the primary immunization, an antigen emulsion prepared with Freund's complete adjuvant or TiterMax Gold adjuvant was administered, with 50-100 μg of antigen injected subcutaneously into each mouse. Booster immunizations were given on days 14 and 28 after the primary immunization, using a mixture of protein and Freund's incomplete adjuvant or... Alum was used to prepare an antigen suspension. Each mouse was injected intraperitoneally or subcutaneously with 25-50 μg of antigen. Blood samples were collected on day 35 to measure antibody titers. Based on the antibody titer, a booster immunization or a final sprint immunization was administered. The sprint immunization involved injecting 50-100 μg of purified protein solution (protein dissolved in phosphate buffer or physiological saline) via the mouse's tail vein. Three days after immunization, the mice were euthanized, and blood, spleen, and lymph node samples were collected for testing and hybridoma preparation.

[0154] 2. Hybridoma preparation

[0155] Hybridoma cells were prepared using methods known to those skilled in the art. Specifically, freshly collected mouse spleen cells or lymph node cells were prepared into a single-cell suspension. Red blood cells were lysed by adding erythrocyte lysis buffer (Beyotime, C3702) and treating at room temperature for 5 minutes. After centrifugation and counting, the cells were mixed with Sp2 / 0 myeloma cell line (spleen cells:sp2 / 0 cells ratio of 2:1), thoroughly washed with 20 mL of electrofusion buffer (BTX cytofusion medium c C), and then electrofused (BTX ECM2001) to prepare hybridoma cells. A 9 mL fusion chamber was used, and the electrofusion parameters were set as follows (AC: 75V / 75V / 30s / 1.0MHz; DC: 800V / 40μs / 1 / 0.000s; AC: 75V / 75V / 30s / 1.0MHz). 7.2 mL of thoroughly mixed cell suspension was added to the fusion chamber, and fusion was initiated immediately. 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 seeded into 96-well cell culture plates (1×10⁻⁶ cells per well). 5 Incubate at 37°C with 5% CO2 (100 μl / well). Change the medium every 3 days. On day 14 post-fusion, perform ELISA to assess cell growth.

[0156] 3. Hybridoma cell screening

[0157] Add 3 μg / mL of antigen protein (100 μL / well) to a 96-well high-adsorption plate (Nunc MaxiSorp). TM (44-2404-21), incubated overnight at 4℃. The next day, block 96-well plates (300 μL / well) with PBS blocking buffer containing 3% skim milk powder, incubate at room temperature for 1 hour, wash 96-well plates 3 times with PBS washing buffer containing 0.1% Tween-20, add 100 μL of hybridoma supernatant to each well, and incubate at room temperature for 1 hour. Wash 96-well plates 3 times, add 100 μL of 2000-fold diluted HRP-labeled goat anti-mouse antibody (Thermo, A10521) to each well, and incubate at room temperature for 1 hour. Wash plates 4 times with washing buffer, add 50 μL of LMB chromogenic solution (Invitrogen, 002023) for 2-5 minutes for color development. Stop the reaction with 1M sulfuric acid, and record the absorbance at 450 nm using a microplate reader. Positive hybridomas are sorted by flow cytometry, and 1 cell / well is seeded into a new 96-well plate, cultured, amplified, and frozen.

[0158] Example 4. Construction and screening of phage display library for anti-human PRLR antibody

[0159] RNA was extracted from immunized mouse spleen cells, and cDNA was synthesized to construct a single-chain antibody (scFv)-phage display library. Target antibodies were then screened using phage surface display technology. Specifically, after cDNA synthesis, PCR amplification was performed using degenerate primers, such as the primer combinations given by AKrebber et al., Journal of Immunological Methods 201 (1997) 35–55, to obtain the heavy and light chain sequences of the target antibody, and a single-chain antibody-phage display immune library was constructed. The target clone sequence was obtained using various panning methods known to those skilled in the art.

[0160] Example 5. Variable region sequences of the heavy and light chains of anti-PRLR antibody

[0161] Table 2 lists the sequence numbers corresponding to the amino acid sequences of the heavy chain, light chain variable region, and CDR region of the anti-PRLR antibody obtained in this disclosure. Sequences prefixed with P are from phage display screening, sequences prefixed with H are from hybridoma screening, and sequences prefixed with HP are from phage libraries constructed using cDNA from positive hybridoma clones.

[0162] Table 2. List of heavy and light chain sequences of murine anti-PRLR antibodies

[0163]

[0164] Example 6. Expression and purification of anti-PRLR antibody

[0165] Primers were designed and PCR amplification was performed to obtain the VH and VL gene fragments of the antibody shown in Table 2. These fragments were then overlapped with the constant region gene fragments CH1-Hinge-CH2-CH3 and CL of the human IgG1 heavy and light chains, respectively, to obtain fusion DNA sequences. These sequences were inserted into the mammalian expression vector pCDNA3.1 to construct the heavy and light chain plasmid vectors expressing the chimeric antibody. After sequencing verification, the plasmid vectors were extracted using an endotoxin-free plasmid extraction kit and stored at -20°C for later use. Expi293F was diluted to a density of approximately 4 x 10⁻⁶. 6Cells / mL. Plasmids expressing the light and heavy chains of antibodies were co-transfected into Expi293F cells using PEI40000 (Polysciences) transfection reagent. Four days later, the cell culture supernatant was collected, centrifuged at high speed, and filtered through a 0.22 μm filter to remove residual cell debris. The filtered cell culture supernatant was purified using a Protein A column. The Protein A column was washed with PBS buffer to remove contaminating proteins. After the A280 reading stabilized at baseline, the target protein was eluted with 0.1 M acetate-sodium acetate solution at pH 3.2. The target protein peak was collected and neutralized with 1 M Tris-HCl solution at pH 8.0. After sample concentration, the sample was further purified using an ENrich™ SEC650 (Bio-red) gel chromatography column to remove aggregates and collect monomer peaks. The collected samples were then analyzed by 4-12% SDS-PAGE gradient gel electrophoresis, aliquoted, and stored at -80℃ for later use.

[0166] Example 7. Species-specific ELISA detection of anti-PRLR antibodies

[0167] Monomeric mouse, monkey, and human PRLR extracellular domain recombinant antigen proteins were diluted in PBS buffer (final concentration 1 μg / mL). The antigen proteins were added to 96-well high-absorption plates (100 μL / well) and incubated overnight at 4°C. The next day, the 96-well plates were blocked with PBS blocking buffer containing 3% skim milk (300 μL / well) and incubated at room temperature for 1 hour. The plates were then washed three times with PBS washing buffer containing 0.1% Tween-20. 100 μL of purified test antibody (10 μg / mL diluted in PBS) was added to each well and incubated at room temperature for 1 hour. The plates were washed three times, and 100 μL of 2000-fold diluted HRP-labeled goat anti-mouse antibody (Thermo) was added to each well and incubated at room temperature for 1 hour. After washing four times with washing buffer, 50 μL of TMB chromogenic solution (Invitrogen) was added for 2-5 minutes for color development. 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 results of the antibodies obtained in this invention. The control protein is a recombinant mesothelin protein with the same tag. As shown in Table 3, all the obtained antibodies specifically bind to the human PRLR extracellular domain. Since the cynomolgus monkey PRLR extracellular domain has a high sequence identity with the human PRLR extracellular domain, differing by only 5 amino acid sites (sequence identity: 97.6%), most of the 23 antibodies, except for antibodies 18, 21, and 23, can bind to the cynomolgus monkey PRLR extracellular domain. Furthermore, antibodies 3, 7, 9, 16, and 19 can also bind to the mouse PRLR extracellular domain.

[0168] Table 3. Species-specific detection of anti-PRLR monoclonal antibodies

[0169]

[0170] Example 7. Affinity detection of anti-PRLR antibody

[0171] The kinetic parameters (kon, koff, and KD) of antibody binding to PRLR ECD were analyzed using biomembrane interferometry (OCTET R2, Sartorius). A protein A sensor ( ProA Biosensors were immersed in PBS buffer containing 0.1% BSA, 0.05% Tween 20, and pH 7.4, with 200 nM of the antibody to be analyzed, allowing the antibody to bind to the sensor surface. After washing, the sensor was sequentially immersed in buffer solutions containing different concentrations of monomeric PRLR antigen, and the affinity parameter KD was obtained by fitting a 1:1 Langmuir model. As shown in Table 4, most antibodies can bind to the target antigen with high affinity, with binding affinity ranging from nM to pM.

[0172] Table 4. Kinetic parameters of binding between anti-PRLR monoclonal antibodies and the extracellular domain of monomeric PRLR

[0173]

[0174]

[0175] Example 10. Humanization of anti-human PRLR monoclonal antibody

[0176] Human antibody sequences with high homology to the target murine antibody sequence were selected as templates through sequence alignment. The CDR region of the murine antibody was determined using the IMGT numbering system. The CDR sequences of the heavy and light chains were transplanted into the human template, while retaining murine amino acids at the following structural positions: Vernier Zone amino acids (Foote et al. J. Mol. Biol, 1992, 224, 487-499); amino acids located at the heavy-light chain interface that affect heavy-light chain pairing 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), thus obtaining the humanized sequence.

[0177] 1. Humanization of P-PR-C05H clone

[0178] Using the common sequence of human antibodies as templates for the heavy and light chains, the mouse CDR region sequence was grafted onto the human template, while retaining mouse amino acids at key structural and functional sites (as shown in Table 5), resulting in the P-PR-C05H-v1 heavy chain (SEQ ID NO: 20) and light chain sequence (SEQ ID NO: 40). The P-PR-C05H-v1 heavy chain sequence was subjected to an S57A mutation to obtain the humanized antibody P-PR-C05H-v2 (heavy chain sequence: SEQ ID NO: 21). The antibody sequences were numbered according to the IMGT numbering rules.

[0179] Table 5. Key mouse amino acid sites retained in P-PR-C05H humanization

[0180]

[0181] 2. Humanization of P-PR-C07H clone

[0182] Using the common sequence of human antibodies as the heavy and light chain templates, the mouse CDR region sequence was grafted onto the human template, while retaining the mouse amino acid sites that are key to structure and function (as shown in Table 6), resulting in the P-PR-C07H-v heavy chain (SEQ ID NO:22) and light chain sequence (SEQ ID NO:40).

[0183] Table 6. Key mouse amino acid sites retained in P-PR-C07H humanization

[0184]

[0185] 3. Humanization of HP-PR01D12 clone

[0186] Using the common sequence of human antibodies as the heavy and light chain templates, the mouse CDR region sequence was grafted onto the human template, while retaining the mouse amino acid sites that are key to structure and function (as shown in Table 7), to obtain the HP-PR01D12-v heavy chain (SEQ ID NO:23) and light chain sequence (SEQ ID NO:41).

[0187] Table 7. Key mouse amino acid sites retained in HP-PR01D12 humanization

[0188]

[0189] Example 11. Affinity detection and epitope analysis of humanized PRLR-specific antibodies

[0190] Biotin-labeled monomeric human or cynomolgus monkey PRLR ECD proteins were used. The labeled proteins were diluted in 100 nM PBS buffer containing 0.1% BSA, 0.05% Tween 20, and pH 7.4. The streptavidin sensor was then immersed in the biotin-labeled protein solution to solidify the target antigen. After washing the sensor with buffer, it was sequentially immersed in buffers containing different concentrations (3.125-100 nM) of humanized antibodies for 2 minutes and 8 minutes for dissociation. Kinetic curves of binding between human PRLR or monkey PRLR ECD proteins were obtained by subtracting from the control sensor. The binding kinetics of the humanized antibodies labeled P-PR-C05H-v2, P-PR-C07H-v, and HP-PR01D12-v were used. The affinity parameter KD was obtained by fitting using a 1:1 Langmuir model. As shown in Table 8, all three humanized antibodies bound human and monkey PRLR ECD proteins with high affinity.

[0191] Table 8. Summary of kinetic and affinity data for PRLR antibodies

[0192]

[0193] The antigen-binding epitopes of three humanized antibodies were analyzed using BLI. A biotin-tagged human monomeric PRLR ECD was immobilized onto a streptavidin (SA) sensor. Antibodies against P-PR-C05H-v2, hP-PR-C07H-v, and HP-PR01D12-v were diluted to a final concentration of 100 nM using PBS buffer containing 0.1% BSA, 0.05% Tween 20, and pH 7.4. The sensor was immersed in the first antibody sample until signal saturation was achieved, followed by immersion in 100 nM of the second antibody. For the first and second antibodies recognizing the same epitope, the second antibody could not bind to the PRLR if the first antibody saturated the epitope on the PRLR surface. When there were overlapping epitopes, the binding signal of the second antibody decreased. For different epitopes, the binding signal of the second antibody remained largely consistent with that of the reference (i.e., the first antibody as the buffer). The epitope distribution was analyzed by the ratio of the binding signal of the second antibody to that of the reference. Among them, a ratio <20% indicates that the two antibodies share the same epitope; a ratio between 20% and 60% indicates partial epitope overlap; and a ratio >60% indicates that the epitopes do not overlap at all. The epitopes of the three humanized antibodies are shown in Table 9. It can be seen that P-PR-C05H-v2 and HP-PR01D12-v bind to the same antigen epitope, while the antigen-binding epitopes of P-PR-C07H-v are close to, but not completely overlapping with, those of P-PR-C05H-v2 and HP-PR01D12-v.

[0194] Table 9. Epitope analysis of humanized antibody binding to human PRLR

[0195] P-PR-C05H-v2 P-PR-C07H-v HP-PR01D12-v P-PR-C05H-v2 11.2％ 35.0％ 15.5％ P-PR-C07H-v 52.7％ 13.4％ 49.8％ HP-PR01D12-v 9.2％ 31.8％ 14.4％

[0196] Example 12. Detection of receptor internalization mediated by humanized PRLR-specific antibody

[0197] The internalization of PRLR by humanized antibodies P-PR-C05H-v2, P-PR-C07H-v, and HP-PR01D12-v was detected by immunofluorescence. T47D cell line (a breast cancer cell line endogenously expressing human PRLR) was co-incubated with 10 μg / mL of humanized antibodies at 4°C for 1 hour. Excess antibody was washed away, and FITC-labeled anti-human Fc antibody was added, followed by incubation at 4°C for another hour. Excess antibody was washed away, and fresh culture medium was added. Control group cells were placed at 4°C, while internalized cells were placed at physiological temperature (37°C, CO2 incubator). After 1 hour, cells were removed, fixed with 4% paraformaldehyde, and the internalization of cell surface antigens was observed under a fluorescence microscope. Figure 1 As shown, antibodies internalized into the cells can be clearly observed after incubation at 37°C for only 1 hour.

[0198] Example 13. Humanized PRLR-specific antibody blocks PRL-mediated PRLR activation and signal transduction.

[0199] The luciferase reporter gene assay was used to determine the blocking effect of humanized PRLR-specific antibody on prolactin (PRL)-mediated receptor dimer conformational changes and downstream signal transduction. Specifically, plasmid pGL4.52 (Promega, #E465A) encoding the STAT5-dependent luciferase reporter gene was transfected into HEK293 cells stably expressing PRLR using Lipofectamine LTX (Thermo, #15338-100). Twenty-four hours after transfection, cells were digested with trypsin, counted, and then indexed at 5 x 10⁻⁶ cells per cell line. 4 Cells were seeded into 96-well white plates at a specific ratio, and then a constant concentration of 5 nM PRL was mixed with different 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, cells were analyzed using Bright-Glo... TMThe activity of luciferase in each well was measured using reagent (Promega, #E2610). The IC50 values ​​for blocking PRL-mediated receptor activation by the humanized antibodies were calculated. As shown in Table 10, P-PR-C05H-v2, P-PR-C07H-v, and HP-PR01D12-v exhibited different abilities to block PRL-mediated receptor activation, with P-PR-C07H-v completely blocking PRL-mediated receptor activation at a concentration of 100 nM (signal value at baseline). Furthermore, P-PR-C05H-v2, P-PR-C07H-v, and HP-PR01D12-v themselves did not activate PRLR at test concentrations ranging from 50 pM to 100 nM.

[0200] Table 10. Humanized antibodies block PRL-mediated receptor activation

[0201]

[0202]

[0203] Example 14. Detection of target cell killing mediated by anti-PRLR antibody-DT3C

[0204] 293T cells and 293T-PRLR cells (5000 cells / 100 μL / well) were seeded into 96-well cell culture plates. After 24 hours of culture, anti-PRLR antibody and DT3C protein were added to fresh culture medium and incubated at 37°C. The final concentrations of anti-PRLR antibody were 0.1, 1, and 10 μg / mL, with corresponding DT3C concentrations of 0.2, 2, and 20 μg / mL. After incubation, the original culture medium in the 96-well plates was aspirated, and the test samples were added to the cells. A culture medium control group and a DT3C control group were also set up. Cells were cultured at 37°C for another 24 hours. After culture, 10 μL of CCK8 solution (purchased from TransGen Biotech, catalog number FC101-02) was added to each well. The 96-well plates were wrapped with aluminum foil and incubated at 37°C for 1 hour. The absorbance at 450 nm was then measured using a Synergy H1 microplate reader (purchased from BioTek). Figure 2 As shown, all three humanized antibodies can effectively mediate the killing of PRLR-positive target cells by DT3C without killing cells that do not express PRLR.

[0205] sequence of this article

[0206]

[0207]

[0208]

Claims

1. An antibody targeting PRLR or an antigen-binding fragment thereof, or a variant having at least 85% sequence identity with said antibody or its antigen-binding fragment and retaining its PRLR binding activity, said antibody targeting the extracellular segment of the prolactin receptor, Preferably, the extracellular segment comprises amino acids 1-210 of SEQ ID NO:99 or 101. More preferably, the antibody comprises: (1) Three HCDRs of the heavy chain variable region shown in any of SEQ ID NO:7, and / or three LCDRs of the light chain variable region shown in any of SEQ ID NO:

29. (2) Three HCDRs of the heavy chain variable region shown in any of SEQ ID NO:8, and / or three LCDRs of the light chain variable region shown in any of SEQ ID NO:28, or (3) Three HCDRs of the heavy chain variable region shown in any of SEQ ID NO:21, and / or three LCDRs of the light chain variable region shown in any of SEQ ID NO:

40.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody's HCDR1 comprises SEQ ID NO: 42 or 43 or 45, or a sequence having at least 85% sequence identity with it, and / or The antibody's HCDR2 comprises SEQ ID NO: 54, 57, or 66, or a sequence having at least 85% sequence identity with it, and / or The antibody's HCDR3 comprises SEQ ID NO: 67, or a sequence having at least 85% sequence identity with it, and / or The antibody's LCDR1 contains SEQ ID NO: 77, or a sequence having at least 85% sequence identity with it, and / or The antibody's LCDR2 comprises SEQ ID NO: 84, or a sequence having at least 85% sequence identity with it, and / or The antibody's LCDR3 comprises SEQ ID NO: 91 or 92, or a sequence having at least 85% sequence identity with it. Preferably, the antibody contains: (1) HCDR1 shown in SEQ ID NO:45, HCDR2 shown in SEQ ID NO:57 and HCDR3 shown in SEQ ID NO:67, and LCDR1 shown in SEQ ID NO:77, LCDR2 shown in SEQ ID NO:84 and LCDR3 shown in SEQ ID NO:92, (2) HCDR1 shown in SEQ ID NO:42, HCDR2 shown in SEQ ID NO:54, and HCDR3 shown in SEQ ID NO:67, and LCDR1 shown in SEQ ID NO:77, LCDR2 shown in SEQ ID NO:84, and LCDR3 shown in SEQ ID NO:91, or (3) HCDR1 shown in SEQ ID NO:43, HCDR2 shown in SEQ ID NO:66 and HCDR3 shown in SEQ ID NO:67, and LCDR1 shown in SEQ ID NO:77, LCDR2 shown in SEQ ID NO:84 and LCDR3 shown in SEQ ID NO:

91.

3. The antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The antibody's VH contains any of the sequences shown in SEQ ID NO:7, 8, and 21, and / or its VL contains any of the sequences shown in SEQ ID NO:29, 28, and 40. Preferably, the antibody contains: (1) VH shown in SEQ ID NO:7 and VL shown in SEQ ID NO:29, (2) VH shown in SEQ ID NO:8 and VL shown in SEQ ID NO:28, (3) VH shown in SEQ ID NO:21 and VL shown in SEQ ID NO:

40.

4. The antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, 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 antibody is a chimeric antibody or a fully human antibody.

5. A fusion protein or antibody conjugate comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4.

6. A polynucleotide selected from: (1) The coding sequence of the antibody or its antigen-binding fragment as described in any one of claims 1-4, or the fusion protein or antibody conjugate as described in claim 5; (2)(1) complementary sequences.

7. A nucleic acid construct expressing the antibody or its antigen-binding fragment as described in any one of claims 1-4, or the fusion protein or antibody conjugate as described in claim 5, or comprising the polynucleotide as described in claim 6. Preferably, the nucleic acid construct is a vector.

8. A phage containing any one of the antibodies or antigen-binding fragments of claims 1-4, or a library containing the phage.

9. A host cell, wherein: (1) Expressing and / or secreting the antibody or antigen-binding fragment thereof as described in any one of claims 1-4; (2) Contains the polynucleotide of claim 6; and / or (3) Contains the nucleic acid construct according to claim 7. Preferably, the host cell is a mammalian cell.

10. A method for generating an antibody or an antigen-binding fragment thereof, comprising: The host cells of claim 9 are cultured under conditions suitable for producing antibodies or antigen-binding fragments thereof, and optionally, the antibodies or antigen-binding fragments thereof are purified from the culture.

11. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-4, a fusion protein, an antibody-drug conjugate, a polynucleotide, a nucleic acid construct, a bacteriophage or a host cell, and a pharmaceutically acceptable excipient.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the fusion protein or antibody conjugate according to claim 5, the polynucleotide according to claim 6, the nucleic acid construct according to claim 7, or the host cell according to claim 9 in the preparation of a medicament for the prevention or treatment of a disease. Preferably, the disease includes: Cancer, endometriosis, hair loss, osteoporosis, obesity, and benign lesions caused by PRLR activation. More preferably, the cancer is a PRLR-related cancer. 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 cancer, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies.

13. A kit for detecting PRLR, used to evaluate the efficacy of drug treatment or diagnose cancer, said kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the fusion protein or antibody conjugate as described in claim 5, the polynucleotide as described in claim 6, the nucleic acid construct as described in claim 7, the bacteriophage as described in claim 8, or the host cell as described in 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-drug conjugate.

14. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the fusion protein or antibody conjugate of claim 5, the polynucleotide of claim 6, the nucleic acid construct of claim 7, the phage of claim 8, or the host cell of claim 9 in the preparation of a kit for detecting PRLR in a sample, evaluating the efficacy of drug treatment, or diagnosing cancer.

Images