Mouse monoclonal antibody specifically combined with PRLR and application thereof

Abstract

The invention provides a mouse monoclonal antibody specifically combined with PRLR and application of the mouse monoclonal antibody. Specifically, the invention provides a PRLR-targeted antibody or an antigen binding fragment thereof, and the antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region. The antibody provided by the invention can specifically recognize and bind to PRLR protein, can mediate internalization after binding to the PRLR protein expressed on the cell surface, and can bind to human and monkey PRLR antigens and inhibit the growth and / or proliferation of tumors or tumor cells.

Description

Technical Field

[0001] This invention relates to the field of antibodies. Specifically, it relates to a mouse monoclonal antibody that specifically binds to PRLR and its applications. Background Technology

[0002] The role of prolactin (PRL) in the pathogenesis of breast cancer is receiving increasing attention. PRL is a protein hormone primarily secreted by eosinophils in the anterior pituitary gland. PRL can stimulate DNA synthesis, epithelial cell proliferation, and breast milk production by influencing primitive cells or neighboring cells and prolactin receptors through autocrine or paracrine mechanisms.

[0003] The prolactin receptor (PRLR) is a member of the cytokine receptor family and mediates the regulation of human breast growth by PRL. In breast cancer research, these data indicate that PRL and its receptor are widely expressed, and in addition to their role in normal differentiation and maturation of the human breast, there is substantial evidence that PRLR is expressed and functions in malignant breast tissue. Immunohistochemical and oncomine analyses both show that PRLR is highly overexpressed in breast cancer tissue (relative to adjacent matched normal tissue).

[0004] Therefore, there is a need in this field to develop an antibody that specifically binds to the PRLR protein. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody that specifically binds to the PRLR protein.

[0006] In a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting PRLR is provided, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region and light chain variable region comprising a complementarity-determining region (CDR) as follows:

[0007] The heavy chain variable region includes the following complementary determinant region (CDR):

[0008] VHCDR1, as shown in SEQ ID NO:6,

[0009] VHCDR2 shown in SEQ ID NO:7, and

[0010] VHCDR3 as shown in SEQ ID NO:8;

[0011] Furthermore, the light chain variable region includes the following complementary determinant region (CDR):

[0012] VLCDR1 shown in SEQ ID NO:2,

[0013] VLCDR2 shown in SEQ ID NO:3, and

[0014] VLCDR3 as shown in SEQ ID NO:4.

[0015] In another preferred embodiment, the CDR sequences of SEQ ID NO:2-4 and SEQ ID NO:6-8 are shown below:

[0016] SEQ ID NO:2: SSIRSTY;

[0017] SEQ ID NO:3: GTS;

[0018] SEQ ID NO:4: QQWSGFPLT;

[0019] SEQ ID NO:6: GYSINSDYA;

[0020] SEQ ID NO:7: ISYSGST;

[0021] SEQ ID NO:8: VRNNWYFDV.

[0022] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:5 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it; and / or the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:1 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it.

[0023] In another preferred embodiment, the antibody has a light chain variable region as shown in SEQ ID NO:1 and a heavy chain variable region as shown in SEQ ID NO:5.

[0024] In another preferred embodiment, SEQ ID NO:1:

[0025] EIVLTQSPALMAASPGEKVTITTCSVSSSIRSTYLHWYQQKSGTSPKPWIYGTSNLASGVPVRFSGSGSGTSFSLTISSMEPEDAATYYCQQWSGFPLTIGAGTKLELK.

[0026] In another preferred embodiment, SEQ ID NO:5:

[0027] DVQLQESGPALIKPSQSLSLTCTVTGYSINSDYAWNWIRQFPGNKLEWMGYISYSGSTDYNPSLESRISITRDTSKNQFFLQLNSVTTEDTATYYCVRNNWYFDVWGAGTTVTVSS.

[0028] In another preferred embodiment, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:9 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it; and / or the amino acid sequence of the light chain is as shown in SEQ ID NO:10 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it.

[0029] In another preferred embodiment, the antibody has a heavy chain as shown in SEQ ID NO:9 and a light chain as shown in SEQ ID NO:10.

[0030] In another preferred embodiment, the antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the three heavy chain CDRs and a heavy chain framework region for connecting the heavy chain CDRs; and the light chain variable region includes the three light chain CDRs and a light chain framework region for connecting the light chain CDRs.

[0031] In another preferred embodiment, the antibody targeting PRLR or its antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody (“scFv”), bisscFv, (scFv)2, microantibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, and disulfide-stabilized Fv protein (“dsFv”).

[0032] In another preferred embodiment, the antibody specifically binds to PRLR or a protein derived therefrom.

[0033] In another preferred embodiment, the antibody is specifically capable of binding to PRLRs derived from humans, mice, and cynomolgus monkeys.

[0034] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0035] In another preferred embodiment, the light chain constant region is of human, mouse, or rabbit origin, preferably of human origin.

[0036] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0037] In another preferred embodiment, the heavy chain constant region is of human, mouse, or rabbit origin, preferably of human origin.

[0038] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0039] In another preferred embodiment, the antibody is a monoclonal antibody.

[0040] In another preferred embodiment, the antibody includes monospecific, bispecific, trispecific, or multispecific antibodies.

[0041] In a second aspect of the invention, a recombinant protein is provided, said recombinant protein having:

[0042] (i) an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0043] (ii) optional tag sequences to assist in expression and / or purification.

[0044] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

[0045] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0046] In another preferred embodiment, the recombinant protein is a fusion protein.

[0047] In another preferred embodiment, the fusion protein is a monospecific antibody (i.e., a monospecific antibody against PRLR), a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0048] In another preferred embodiment, the bispecific or multispecific antibody not only resists PRLR, but also specifically binds to additional target antigens (such as other tumor antigens, such as other antigens of gastric cancer or other tumor antigens).

[0049] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0050] In another preferred embodiment, the recombinant protein further includes an additional fusion element (or fusion polypeptide fragment) fused together with said element (i).

[0051] In a third aspect of the invention, a chimeric antigen receptor (CAR) is provided, wherein the antigen-binding domain of the chimeric antigen receptor contains an antibody single-chain variable region sequence scFv targeting PRLR, and the heavy chain variable region and light chain variable region of the scFv include complementarity-determining regions (CDRs) as follows:

[0052] The heavy chain variable region includes the following complementary determinant region (CDR):

[0053] VHCDR1, as shown in SEQ ID NO:6,

[0054] VHCDR2 shown in SEQ ID NO:7, and

[0055] VHCDR3 as shown in SEQ ID NO:8;

[0056] Furthermore, the light chain variable region includes the following complementary determinant region (CDR):

[0057] VLCDR1 shown in SEQ ID NO:2,

[0058] VLCDR2 shown in SEQ ID NO:3, and

[0059] VLCDR3 as shown in SEQ ID NO:4.

[0060] In a fourth aspect of the invention, a polynucleotide is provided that encodes an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, or a chimeric antigen receptor (CAR) as described in the third aspect of the invention.

[0061] In a fifth aspect of the invention, a carrier is provided, said carrier containing the polynucleotide described in the fourth aspect of the invention.

[0062] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0063] In another preferred embodiment, the vector is a retroviral vector.

[0064] In a sixth aspect of the invention, a host cell is provided, wherein the host cell contains an exogenous polynucleotide as described in the fourth aspect of the invention integrated into a vector or chromosome as described in the fifth aspect of the invention.

[0065] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0066] In another preferred embodiment, the cell is a mammalian cell.

[0067] In another preferred embodiment, the cells are NK cells or T cells.

[0068] In another preferred embodiment, the host cell is an engineered immune cell.

[0069] In a seventh aspect of the invention, a method for preparing CAR-NK cells or CAR-T cells, wherein the CAR-NK cells or CAR-T cells express the chimeric antigen receptor described in the third aspect of the invention, includes the following steps:

[0070] The polynucleotide described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention is introduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

[0071] In an eighth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a chimeric antigen receptor as described in the third aspect of the invention, a polynucleotide as described in the fourth aspect of the invention, a carrier as described in the fifth aspect of the invention, or a host cell as described in the sixth aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0072] In another preferred embodiment, the pharmaceutical composition is a formulation, preferably a liquid formulation.

[0073] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0074] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a host cell or a combination thereof as described in the sixth aspect of the invention, and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentages are percentages by mass of the pharmaceutical composition.

[0075] In another preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of cancer or tumors.

[0076] In another preferred embodiment, the tumor is selected from: gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.

[0077] In another preferred embodiment, the tumor is selected from: ductal carcinoma of the breast, adenocarcinoma of the breast, and liver cancer.

[0078] In a ninth aspect of the present invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0079] (a) An antibody portion, said antibody portion being selected from the group consisting of: antibodies or antigen-binding fragments thereof as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, or combinations thereof; and

[0080] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0081] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0082] In a tenth aspect of the invention, the use of an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a chimeric antigen receptor as described in the third aspect of the invention, a polynucleotide as described in the fourth aspect of the invention, a vector as described in the fifth aspect of the invention, a host cell as described in the sixth aspect of the invention, a pharmaceutical composition as described in the eighth aspect of the invention, or an immunoconjugate as described in the ninth aspect of the invention is provided.

[0083] (a) Preparation of detection reagents or kits; and / or

[0084] (b) To prepare medicines or formulations for the prevention and / or treatment of PRLR-related diseases.

[0085] In another preferred embodiment, the PRLR-related disease is cancer or tumor.

[0086] In another preferred embodiment, the tumor is selected from: gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.

[0087] In an eleventh aspect of the present invention, a method for in vitro detection of PRLR protein in samples (including diagnostic or non-diagnostic samples) is provided, the method comprising the steps of:

[0088] (1) Contact the sample with the antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention;

[0089] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of PRLR protein in the sample.

[0090] In another preferred embodiment, the detection is for in vitro, non-therapeutic, and non-diagnostic purposes.

[0091] In another preferred embodiment, the method is an immunochemical cell (ICC) detection method, or an immunohistochemical (IHC) detection method, or a whole-cell ELISA detection method, or a cell lysate ELISA detection method.

[0092] In a twelfth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0093] (a) Culturing the host cells described in the sixth aspect of the present invention under suitable expression conditions;

[0094] (b) Isolate a recombinant polypeptide from the culture, wherein the recombinant polypeptide is an antibody or its antigen-binding fragment as described in the first aspect of the present invention, or a recombinant protein as described in the second aspect of the present invention.

[0095] In a thirteenth aspect of the present invention, a detection plate is provided, the detection plate comprising: a substrate (support plate) and a test strip, the test strip containing an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, an immunoconjugate as described in the ninth aspect of the present invention, or a combination thereof.

[0096] In a fourteenth aspect of the invention, a kit is provided, the kit comprising:

[0097] (1) A first container containing the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; and / or

[0098] (2) A second container containing a secondary antibody against the antibody;

[0099] Alternatively, the kit may contain the detection plate described in aspect thirteen of the present invention.

[0100] In a fifteenth aspect of the invention, a method for treating a disease associated with abnormal PRLR expression or function is provided, comprising administering to a subject requiring treatment an effective amount of an antibody or antigen-binding fragment thereof as described in a first aspect of the invention, a recombinant protein as described in a second aspect of the invention, a host cell as described in a sixth aspect of the invention, or a pharmaceutical composition as described in an eighth aspect of the invention.

[0101] In another preferred embodiment, the disease associated with abnormal PRLR expression or function is a tumor or cancer.

[0102] In a sixteenth aspect of the invention, the use of the antibody or antigen-binding fragment thereof described in the first aspect of the invention, or the recombinant protein described in the second aspect of the invention, or the host cell described in the sixth aspect of the invention, and / or the pharmaceutical composition described in the eighth aspect of the invention, in the preparation of a medicament for treating diseases associated with abnormal PRLR expression or function is provided.

[0103] In another preferred embodiment, the PRLR expression abnormality refers to PRLR overexpression.

[0104] In another preferred embodiment, the overexpression refers to the ratio of the expression level (F1) of PRLR to the expression level (F0) under physiological conditions (i.e., F1 / F0) being ≥1.5, preferably ≥2, and more preferably ≥2.5.

[0105] In another preferred embodiment, the drug is used for the prevention and / or treatment of tumors.

[0106] In another preferred embodiment, the drug is used to prevent and / or treat tumor occurrence, growth and / or metastasis.

[0107] In another preferred embodiment, the tumor includes solid tumors and hematologic malignancies.

[0108] In another preferred embodiment, the tumor is a tumor that highly expresses PRLR.

[0109] In another preferred embodiment, the tumor is selected from: gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.

[0110] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0111] Figure 1 The results of the IF and FACS experiments on the anti-human-monkey cross-overexpression of the monoclonal antibody described in this invention are shown; where CL885 represents the #885 antibody; Figure A shows the immunofluorescence assay (IF) detection of human-monkey cross-overexpression of PRLR, where red fluorescence represents the specificity of the anti-PRLR antibody as red monoclonal antibody staining for PRLR overexpression in 293T cells, and green fluorescence represents the immunofluorescence colocalization of the green GFP-labeled target in 293T cells; Figure B shows the FACS detection results of human-monkey cross-overexpression of PRLR.

[0112] Figure 2 The results of FACS assays show the binding activity of the PRLR chimeric antibody ch885 to PRLR in different cells; Figure A represents T47D cells, Figure B represents Huh-7 cells; h16f represents the antibody disclosed in the existing patent as a positive control, and HuIgG represents the negative control antibody.

[0113] Figure 3This shows the results of an ELISA assay to detect the binding activity of the PRLR chimeric antibody ch885 to PRLR; where #885 antibody represents the half-maximal effective concentration of human PRLR antigen in vitro; h16f represents a chimeric antibody disclosed in a prior patent, used as a positive control.

[0114] Figure 4 The results show the tumor cell killing effect of the PRLR chimeric antibody ch885; T47D in the figure are PRLR-high expressing cells (i.e., positive cell lines), and MDA-MB-231 are PRLR-low expressing cells (i.e., negative cell lines). Detailed Implementation

[0115] Through extensive and in-depth research, the inventors unexpectedly discovered a class of monoclonal antibodies against PRLR for the first time. The antibodies provided by this invention can specifically recognize and bind to the PRLR protein, and mediate internalization after binding to PRLR protein expressed on the cell surface. The antibodies of this invention exhibit excellent binding activity against PRLR from multiple species (especially human and monkey). Furthermore, the antibodies of this invention can also mediate the specific killing of PRLR-overexpressing cancer cells by immune cells through ADCC. Based on these findings, this invention was completed.

[0116] the term

[0117] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0118] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0119] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0120] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, comprising the antibody against PRLR and compositions thereof of the present invention, to a patient having one or more disease symptoms, for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is given an amount of the therapeutic agent that effectively relieves one or more disease symptoms (therapeutic effective amount).

[0121] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur but are not required to occur.

[0122] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0123] As used herein, the term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0124] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0125] As used herein, the terms “antibody of the present invention”, “PRLR antibody”, “#885 antibody”, “CL885”, and “CL070885” are used interchangeably and all refer to the monoclonal antibody numbered 885.

[0126] As used in this article, the terms "PRLR chimeric antibody" and "ch885" are used interchangeably, both referring to the antibody variable region sequence cloned into a vector containing a human Fc fragment, transfected into 293F cells for antibody expression, and then further purified by Protein A to obtain the chimeric antibody.

[0127] As used herein, the term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or their antigen-binding portions. Each H chain contains a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0128] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0129] As used in this article, the terms “light chain variable zone” and “VL” are used interchangeably.

[0130] As used herein, the term "antigen-binding domain" and the like encompasses any naturally occurring, enzymatically available, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains, can be used, for example, deriving antigen-binding fragments of antibodies from intact antibody molecules. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, such as to arrange one or more variable domains and / or constant domains into a suitable layout, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0131] As used herein, non-limiting examples of antigen-binding fragments or antigen-binding domains include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., independent complementarity-determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides.

[0132] As used herein, antigen-binding fragments or antigen-binding domains will generally contain at least one variable domain. The variable domain can have any size or amino acid composition and will typically contain at least one CDR adjacent to or conforming to one or more frame sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be arranged opposite each other in any suitable configuration. For example, the variable region can be a dimer and contain VH-VH, VH-VL, or VL-VL dimers. Optionally, the antigen-binding domain can contain monomeric VH or VL domains.

[0133] In a given antibody's light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia based on the antibody's three-dimensional structure and the topology of the CDR loop; Kabat (Kabat, E., et al., USDapartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); the International Immuno GeneTics database (IMGT); the EU numbering system; and Chothia definitions based on loop structural positions.

[0134] It should be understood that the precise amino acid sequence boundaries of the CDR in this invention can optionally be defined using the different assignment systems mentioned above. Preferably, unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system.

[0135] PRLR

[0136] PRLR is a gene encoding the receptor for the anterior pituitary hormone prolactin, belonging to the type I cytokine receptor family. Prolactin-dependent signaling is a result of prolactin (ligand)-induced dimerization of the prolactin receptor. Several alternatively spliced ​​transcript variants of this gene, encoding different membrane-bound and soluble subtypes, have been described, potentially playing a role in regulating the endocrine and autocrine effects of prolactin in normal tissues and cancer. Prolactin (PRL) is a protein hormone secreted by the pituitary gland, primarily promoting mammary gland growth and stimulating and maintaining lactation. Previous studies have shown that high PRL levels increase the risk of invasive breast cancer in women. Elevated expression levels of PRL and PRLR are observed in most ER breast cancer cells and tissues, and in ER tumors. PRL activates downstream signaling pathways and influences endocrine therapy resistance by binding to the prolactin receptor (PRLR). Research on PRL and its receptor will contribute to exploring ways to reverse endocrine therapy resistance in breast cancer.

[0137] The role of prolactin (PRL) in the pathogenesis of breast cancer is receiving increasing attention. PRL is a protein hormone primarily secreted by eosinophils in the anterior pituitary gland. PRL can stimulate DNA synthesis, epithelial cell proliferation, and breast milk production by influencing primitive or neighboring cells and prolactin receptors through autocrine or paracrine mechanisms. PRLR is a member of the cytokine receptor family and mediates the regulation of human breast growth. In breast cancer research, these data indicate that PRL and its receptor are widely expressed, and in addition to their role in normal differentiation and maturation of the human breast, there is substantial evidence that PRLR is expressed and functions in malignant breast tissue. Immunohistochemistry and oncomine analysis both show that PRLR is highly overexpressed in breast cancer tissue (relative to adjacent matched normal tissue).

[0138] Antibody

[0139] As used herein, the terms "antibody" or "immunoglobulin" refer to heterotetraglycoproteins of approximately 150,000 Daltons with identical structural features, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VH) at one end. L The light chain has a constant region at one end and a variable region at the other. The constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specialized amino acid residues form an interface between the variable regions of the light and heavy chains.

[0140] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0141] This invention includes not only complete monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0142] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., 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 fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0143] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0144] The present invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0145] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0146] Table A

[0147]

[0148]

[0149] As used in this article, the terms “ADCC” or “antibody-dependent cell-mediated cytotoxicity” include cell-mediated reactions in which nonspecific cytotoxic cells expressing FcγR recognize antibodies bound to target cells, causing target cell lysis. In various contexts, enhanced ADCC effector function can refer to enhanced titer or enhanced efficacy. In the experimental context, “titer” refers to the concentration of the antibody (half-maximum effective concentration) at which a specific therapeutic efficacy EC50 is observed. In the experimental context, “efficacy” refers to the maximum possible effector function of an antibody at saturation levels.

[0150] Preferably, the antibody described herein is one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibody can be developed using various methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0151] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0152] The antibody of the present invention can be a double-chain or single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0153] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0154] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids.

[0155] The animal is preferably a mammal, such as a mouse.

[0156] The antibodies of this invention may be chimeric antibodies, humanized antibodies, CDR-grafted and / or modified antibodies that target PRLR (e.g., human PRLR, mouse PRLR or cynomolgus monkey PRLR).

[0157] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0158] In the above-described content of the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids can be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2.

[0159] Recombinant protein

[0160] The present invention also provides a recombinant protein comprising one or more of the heavy chain CDR1 (VHCDR1), heavy chain CDR2 (VHCDR2), and heavy chain CDR3 (VHCDR3) of the antibody of the present invention, and / or one or more of the light chain CDR1 (VLCDR1), light chain CDR2 (VLCDR2), and light chain CDR3 (VLCDR3) of the antibody of the present invention.

[0161] Preferably, the recombinant protein further includes an antibody heavy chain constant region and / or an antibody light chain constant region. The antibody heavy chain constant region is conventional in the art, preferably a rat-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region, and more preferably a human-derived antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat-derived antibody light chain constant region or a human-derived antibody light chain constant region, and more preferably a human-derived antibody light chain constant region.

[0162] In another preferred embodiment, the recombinant protein comprises the antibody of the present invention.

[0163] The recombinant protein is a conventional protein in the art, preferably one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies.

[0164] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids.

[0165] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, comprising an Fd segment of a light chain variable region, a light chain constant region, and a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0166] The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0167] The single-region antibody described is a conventional single-region antibody in the art, which includes only the heavy chain variable region.

[0168] The recombinant protein is prepared using conventional methods in the art. Preferably, the preparation method involves isolating the protein from an expression transformant or obtaining it through artificial synthesis of the protein sequence. The method for isolating the protein from the expression transformant is preferably as follows: cloning a polynucleotide molecule encoding the protein and carrying a point mutation into a recombinant vector; transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant; and culturing the obtained recombinant expression transformant to isolate and purify the recombinant protein.

[0169] Polynucleotides

[0170] The present invention also provides a polynucleotide encoding the above-described antibody or recombinant protein of the present invention or a chimeric antigen receptor (CAR) construct of the antibody of the present invention.

[0171] The preparation method of the polynucleotide is a conventional preparation method in the art. Preferably, it includes the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full-sequence synthesis.

[0172] Those skilled in the art will understand that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homologue. The polynucleotide homologue of this invention can be prepared by substituting, deleting, or adding one or more bases of the gene encoding the protein sequence, while maintaining antibody activity.

[0173] carrier

[0174] The present invention also provides a recombinant expression vector containing the nucleic acid.

[0175] The recombinant expression vector described herein can be obtained by conventional methods in the art, namely, by linking the nucleic acid molecule described in this invention to various expression vectors. The expression vector can be any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule. Preferably, the vector includes various plasmids, granules, bacteriophages, or viral vectors, etc.

[0176] The present invention also provides a recombinant expression transformant comprising the above-described recombinant expression vector.

[0177] The recombinant expression transformant is prepared using conventional methods in the art, preferably by transforming the recombinant expression vector into host cells. The host cells can be any common host cells in the art, as long as they allow the recombinant expression vector to replicate stably and effectively express the carried nucleic acid. Preferably, the host cells are E. coli TG1 or E. coli BL21 cells (expressing single-chain antibodies or Fab antibodies), or HEK293 or CHO cells (expressing full-length IgG antibodies). Transforming the aforementioned recombinant expression plasmid into host cells yields the preferred recombinant expression transformant of this invention. The transformation method is a conventional method in the art, preferably chemical transformation, heat shock, or electroporation.

[0178] Antibody preparation

[0179] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0180] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0181] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0182] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0183] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0184] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0185] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0186] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0187] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0188] Antibody-drug conjugates (ADCs)

[0189] As used herein, the terms “antibody-drug conjugate,” “ADC,” and “antibody-drug conjugate” are used interchangeably and all refer to antibody-drug conjugates (ADCs) based on the antibodies of this invention.

[0190] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0191] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0192] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0193] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0194] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0195] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0196] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0197] In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0198] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0199] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0200] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0201] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula:

[0202]

[0203] in:

[0204] Ab is an antibody.

[0205] LU stands for connector;

[0206] D is a drug;

[0207] Furthermore, the subscript p is a value selected from 1 to 8.

[0208] Methylaurestatin E (MMAE)

[0209] Methylaurestatin E (MMAE) is an antimitotic agent that inhibits cell division by blocking microtubule polymerization, and is of considerable interest among antibody-drug conjugates (ADCs). MMAE is derived from a polypeptide called dolastatin found in the marine shell-less mollusc Dolabella auricularia. These polypeptides have shown effective in vitro and in vivo activity against various lymphomas, leukemias, and solid tumors in preclinical studies. CAS RN: 474645-27-7; Molecular formula: C 39 H 67 N5O7; Molecular weight: 717.97858; The structural formula of MMAE is shown below:

[0210]

[0211] VcMMAE(mc-vc-PAB-MMAE)

[0212] VcMMAE is part of an antibody-coupled device (ADC) and possesses anticancer activity. It consists of MMAE (a tubulin inhibitor) and valine-citrulline (VC). The structural formula of VcMMAE is shown below:

[0213]

[0214] application

[0215] The present invention also provides the use of the antibodies, recombinant proteins, chimeric antigen receptor (CAR) constructs and / or immune cells of the present invention, for example, for the preparation of diagnostic agents or pharmaceuticals.

[0216] Preferably, the drug is a drug for the prevention and / or treatment of diseases associated with abnormal PRLR expression or function.

[0217] In this invention, the diseases associated with abnormal PRLR expression or function are those conventionally associated with abnormal PRLR expression or function in the art. Preferably, the diseases associated with abnormal PRLR expression or function are cancer.

[0218] In this invention, the cancer is a conventional cancer in the art, preferably gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, bile duct cancer, lung cancer, or non-small cell lung cancer.

[0219] Detection uses and kits

[0220] The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0221] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include, for example, resected tumor samples, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

[0222] The samples used in this invention include fixed or preserved cell or tissue samples.

[0223] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0224] Pharmaceutical Composition

[0225] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0226] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0227] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0228] The pharmaceutical composition described in this invention is a pharmaceutical composition for the prevention and / or treatment of diseases associated with abnormal PRLR expression or function.

[0229] The pharmaceutical composition of the present invention can be directly used to bind PRLR protein molecules, and therefore can be used for the prevention and treatment of diseases such as tumors.

[0230] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0231] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0232] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0233] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0234] This invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of diseases associated with abnormal PRLR expression or function. Preferably, the disease associated with abnormal PRLR expression or function is a tumor / cancer.

[0235] The main advantages of this invention include:

[0236] (1) The antibody or its antigen-binding fragment of the present invention can specifically recognize PRLR protein expressed on the cell surface;

[0237] (2) The antibody or its antigen-binding fragment of the present invention can be internalized by cells, especially tumor cells;

[0238] (3) The antibody or its antigen-binding fragment of the present invention can be used to construct an immunoconjugate (ADC molecule), and the immunoconjugate can also inhibit the growth and / or proliferation of tumors or tumor cells;

[0239] (4) The human-mouse chimeric antibody of the present invention can bind to human and monkey PRLR antigens.

[0240] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0241] Experimental materials

[0242] The h16f antibody is a patented antibody, and its sequence is obtained and then expressed by Abimate. The VH and VL sequences of the h16f patented antibody are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0243] Example 1: Preparation of mouse monoclonal antibody hybridoma cells containing PRLR protein

[0244] 1.1 Preparation of antigen

[0245] The extracellular polypeptide fragment (Ala 221-234Asp) of the PRLR region was used as the immunogen. This region is identical in both humans and mice. The antigen was purchased from Kaijing Biotechnology.

[0246] 1.2 Immunization of mice

[0247] Each antigen will be used to immunize 12 Balb / c mice (8-12 weeks old), and their serum titers will be monitored to determine the optimal number of immunizations. Optimized adjuvants and immunization methods generate high-affinity antibodies (IgG subtype) against most antigenic peptides. After the primary immunization, there will be 3 to 4 booster immunizations. After each booster, mouse serum titers will be measured (recombinant protein coated as anti-antigen). Mice with acceptable titers will be given one dose and used for fusion, while mice with unacceptable titers will receive one or two more boosters until the highest titer is achieved, at which point fusion will take place.

[0248] 1.3 Serum detection and screening

[0249] Blood was collected from the orbital sinus of immunized mice, and serum titers were detected by ELISA (recombinant protein was used as the antigen coating). The serum titer must be greater than 10K; otherwise, booster immunization should be performed.

[0250] 1.4 Fusion and Screening

[0251] The entire spleen and half of the lymph nodes were harvested and fused with myeloma SP2 / 0 cell lines. The fusion process used was an optimized PEG fusion method. The fused cells were seeded onto four 384-well plates (10 cells per well). 2 Up to 10 4 Cells were cultured. Supernatants from all wells were collected, and positive cell lines were screened by detecting the reaction of the cell supernatants to the PRLR recombinant protein using ELISA. Positive wells containing cells were transferred to 96-well plates for further culture. After several days of growth, supernatants from all wells were collected, and the reactivity of the cell supernatants to the PRLR recombinant protein was detected by ELISA. The binding affinity of different dilutions of cell supernatants to the PRLR recombinant protein in the positive wells was further analyzed to rank the cell lines by affinity. The 60 parental clones with the highest affinity were selected for subcloning.

[0252] 1.5 Subcloning and Screening

[0253] Subcloning was performed using limiting dilution and ELISA to obtain monoclonal hybridoma cells. Cells were seeded into 96-well plates and cultured until approximately 1 / 6 of the bottom was covered. ELISA was used to detect the reaction of the supernatant in each well to the PRLR recombinant protein; two wells with high OD values ​​and good cell condition were selected for the next round of subcloning. This process was repeated until 100% of the cells in the wells were positive. At this point, monoclonal cell lines were obtained. After the final round of subcloning, all positive cells (hybridoma cells) were immediately expanded into larger cultures; some were cryopreserved for later use, and the rest were used to prepare ascites fluid. Through this selection process, several high-performance antibodies were obtained.

[0254] 1.6 Ascites preparation and antibody purification

[0255] The obtained monoclonal cell lines were injected into F1 mice via abdominal injection for antibody production. The resulting ascites fluid was purified with Protein A / G and used for subsequent assays.

[0256] Example 2: Antibody specifically recognizes PRLR antigen expressed on cell surface

[0257] 2.1 Plasmid Preparation

[0258] The plasmid was constructed by Sangon Biotech through whole-genome synthesis and transfected into E. coli (TOP10 strain). It was cultured overnight at 37°C, and plasmid was extracted using a kit (MN NucleoBond Xtra Midi Ultra-Rapid Transfection Grade Plasmid Extraction Kit 740410.50 from Germany).

[0259] 2.2 Transfection

[0260] Thoroughly aspirate the culture medium from 293T cells. Add an appropriate amount of PBS to a culture dish, shake the dish, discard the PBS, and repeat 2-3 times until the washed PBS is colorless. Aspirate the PBS, add an appropriate amount of trypsin, and place in a 37°C incubator until the cells are digested into single cells. Add 5 mL of complete culture medium to each culture dish to stop the digestion. Collect the single-cell suspension into a 50 mL centrifuge tube, centrifuge at 1000-1500 rpm for 5 min, discard the supernatant to obtain the cell pellet. Resuspend the cells in complete culture medium, count the cells, and then dilute the cells with complete culture medium according to the required cell volume and plate them. Incubate overnight in a 37°C CO2 incubator. Remove the plasmid from -20°C, thaw it, and mix it thoroughly for later use. Incubate OPTI-MEM and serum-free culture medium in a 37°C water bath. Remove the prepared PEI solution (1 mg / mL, ddH2O / PBS, sterilized by filtration through a 0.2 μm membrane, stored at 4°C) from a 4°C freezer, invert several times to mix thoroughly.

[0261] Transfection system configuration:

[0262] Solution A (plasmid + OPTI-MEM): Add 30 μg of plasmid + 1 mL of OPTI-MEM to each 10 cm culture dish.

[0263] Solution B (PEI+OPTI-MEM): Add 60μL PEI+1mL OPTI-MEM to each 10cm culture dish.

[0264] The plasmid to PEI mass ratio is 1:3. Let it stand at room temperature for 5 minutes. After standing, add solution B to solution A and mix well. Let it stand at room temperature for 20 minutes. During the standing period, aspirate the culture medium from the 6-well plate or 10cm culture dish and slowly add 1.5mL (per well of a 6-well plate) or serum-free culture medium.

[0265] After the transfection system has settled, use a pipette to add the transfection system to a 6-well plate or culture dish, shake the dish to mix the transfection system with serum-free medium. After transfection, incubate the cells in a 37°C CO2 incubator for 4–6 hours, then change the medium to complete medium and incubate overnight.

[0266] The transfected 293T cells were observed using a fluorescence microscope with 488 channels. The presence of green fluorescence in the cytoplasm indicated successful transfection. The results showed that the transfected 293T cells expressed either monkey-derived or human-derived PRLR antigens.

[0267] 2.3 Antibody specifically recognizes PRLR antigen expressed on the cell surface

[0268] Add 8% paraformaldehyde (8% PFA) solution to the 96-well cell culture plates containing overexpressing cells and fix at room temperature for 15 min to ensure adequate cell fixation. Discard the paraformaldehyde. Wash four times with 100 μL PBS. Add 100 μL of cell lysis blocking buffer (PBS solution containing 10% goat serum and 0.1% Triton 100) to each well after washing and block overnight.

[0269] Add the primary antibody (purified PRLR antibody, e.g., CL885), mix gently, and incubate at room temperature for 3 hours. After primary antibody incubation, discard the supernatant and wash each well with 100 μL of PBS for a total of 6 washes. Be careful to insert the pipette tip into the well along the wall and aspirate from the bottom corner of the plate. Select the goat anti-mouse fluorescently labeled secondary antibody (Alexa Fluor 594 AffiniPure Goat Anti-Mouse IgG (H+L), purchased from Jackson) according to the task sheet. Prepare a secondary antibody + DAPI dilution buffer using PBS containing 10% goat serum, diluting the secondary antibody 1:400 and DAPI 1:10000. Add 100 μL of the prepared secondary antibody + DAPI dilution buffer to each well and incubate at room temperature in the dark for 45 minutes. After secondary antibody incubation, discard the supernatant and wash each well with 100 μL of PBS for a total of 3 washes. After washing, place the cells in PBS and observe the results under a fluorescence microscope.

[0270] The results are as follows Figure 1 As shown in AB, the monoclonal antibody (CL885) produced by the #885 cell line can not only recognize human PRLR antigens, but also specifically recognize monkey PRLR antigens; it has excellent human-monkey cross-reactivity; and it is helpful to use monkeys as a model for performance verification when developing antibody drugs.

[0271] Example 3: FACS Validation of Endogenous PRLR Antibody in Ascites Fluid

[0272] MB-231 and T47D cells were evenly seeded into 96-well plates and cultured overnight. The 96-well cell culture plates were removed from the incubator, and without discarding the culture medium, 100 μL of 8% paraformaldehyde (8% PFA) solution was slowly added directly to each well. The plates were fixed at room temperature for 15 min to allow the paraformaldehyde to fully fix the cells. The paraformaldehyde was then discarded using a pipette. The cells were washed four times with 100 μL of PBS. After washing, 100 μL of cell-blocking buffer (PBS solution containing 10% goat serum and 0.1% Triton 100) was added to each well, and the plates were blocked overnight.

[0273] Primary antibody (CL885 or control IgG antibody) was diluted to a final concentration of 10 μg / mL and incubated at 4℃ for 1 h. After incubation, the supernatant was discarded, and each well was washed with 100 μL of PBS for a total of 6 washes. Goat anti-mouse fluorescently labeled secondary antibody (Alexa Fluor488 Affini Pure Goat Anti-Mouse IgG (H+L), purchased from Jackson) was used. A mixed dilution of secondary antibody and DAPI was prepared using PBS containing 10% goat serum, with the secondary antibody diluted 1:400 and DAPI diluted 1:10000. 100 μL of the prepared mixed dilution was added to each well, and the mixture was incubated at room temperature in the dark for 45 min. After secondary antibody incubation, the supernatant was discarded, and each well was washed with 100 μL of PBS for a total of 3 washes. Fluorescence intensity was detected by flow cytometry. The results are shown in Table 1.

[0274] Table 1

[0275]

[0276] Note: MB-231(488) represents PRLR negative cell line, T47D(488) represents PRLR positive cell line; CL070885 represents #885 antibody; (488) represents fluorescence intensity detected at 488nm excitation wavelength, and RMFI represents relative MFI with IgG.

[0277] The results in Table 1 show that, according to the FACS test, the anti-PRLR antibody (#885) can bind to T47D cells (i.e., PRLR-positive cell lines) and does not selectively bind to MB-231, indicating that the #885 antibody can specifically recognize PRLR antibodies on the cell surface.

[0278] Example 4: Antibody Variable Region Sequencing

[0279] Hybridoma cells containing the #885 antibody were expanded and cultured. After Trizol lysis, cellular RNA was extracted and reverse transcribed to obtain cDNA. The variable region of the antibody was amplified using specific degenerate primers and cloned into a T-plasmid. Sequencing was performed using SP6 primers.

[0280] The amino acid sequence of the antibody light chain variable region VL is as shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:5.

[0281] Example 5: FACS detection of PRLR chimeric antibody binding activity to PRLR

[0282] 5.1 Preparation of PRLR chimeric antibody

[0283] The antibody variable region sequence obtained from sequencing in Example 4 was cloned into a vector containing the human Fc fragment, transfected into 293F cells for antibody expression, and then further purified by Protein A to obtain the chimeric antibody ch885.

[0284] 5.2 Combination activity test

[0285] Discard or thoroughly aspirate the culture medium from two cell lines that highly express PRLR: T47D (human breast duct carcinoma cells) and Huh-7 (human liver carcinoma cells). Wash 2–3 times with PBS until the PBS is colorless. Digest the cells with EDTA, thoroughly aspirating any remaining PBS, then add 1 mM EDTA according to the size of the culture dish. Continue digestion until single cells are formed. Add 5 mL of PBS to each culture dish to stop digestion. Collect the single-cell suspension in a 50 mL centrifuge tube, centrifuge at 1000–1500 rpm for 5 min, and discard the supernatant to obtain the cell pellet. Resuspend the cell pellet in 10–20 mL of PBS, gently pipetting 2–3 times, centrifuge at 1000–1500 rpm for 5 min, and discard the PBS. Resuspend the cells in blocking buffer and incubate at 4°C for 30 min. Seed the cells into 50 μL of U-bottom 96-well plates. Pour the prepared ch885, h16f, and HuIgG primary antibodies into the sample well, gently mix with a pipette 2-3 times, and add 50 μL to each well of the cell sample plate. Incubate at 4°C for 1 h. After incubation, centrifuge the plate in a 96-well centrifuge at 1000-1500 rpm, discard the supernatant, wash 2-3 times with PBS, centrifuge again, and incubate with secondary antibody at 4°C in the dark for 45 min. Wash 3 times with PBS after incubation. Detect cell fluorescence intensity using flow cytometry, record and save the median fluorescence value.

[0286] The results are as follows Figure 2 As shown, the PRLR chimeric antibody ch885 can recognize tumor cells T47D and Huh-7, which highly express PRLR. Figure 2 (A and 2B) and its binding activity is superior to that of the antibody h16f disclosed in existing patents. Especially in T47D cells, the binding activity of the PRLR chimeric antibody ch885 is tens of times higher than that of the antibody h16f disclosed in existing patents.

[0287] Example 6: ELISA detection of PRLR chimeric antibody binding activity to PRLR

[0288] Recombinant protein PRLR was coated onto ELISA strips at a concentration of 1 μg / mL, 0.1 mL / well, and incubated overnight at 4°C. After washing with PBST, 10% fetal bovine serum was added, and the strips were blocked at 37°C for 1 hour. Different dilutions of PRLR chimeric antibodies ch885, h16f, and HuIgG were added, and the strips were reacted at 37°C for 1 hour. After washing with PBST, horseradish peroxidase-labeled goat anti-human IgG secondary antibody (Goatanti-Mouse (HRP), ThermoFisher Scientific) was added, and the strips were reacted at 37°C for 30 minutes. The strips were washed five times with PBST, and residual droplets were patted dry on absorbent paper. 100 μL of TMB (eBioscience) was added to each well, and the strips were incubated at room temperature (20±5°C) in the dark for 1.5 minutes. 100 μL of 2M H2SO4 stop solution was added to each well to stop the substrate reaction. The OD value was read at 450 nm using a microplate reader to analyze the binding ability of the antibody to the target antigen PRLR. The results are shown in Table 2 and [Table data would be inserted here]. Figure 3 As shown in Table 2, the antibody concentration unit is ng / mL. The antibody binding capacity results, after conversion to nanomolar concentration, are as follows: Figure 3 As shown:

[0289] Table 2

[0290]

[0291] Note: h16f represents the chimeric antibody disclosed in the existing patent, i.e., the positive control, and HuIgG represents the negative control antibody.

[0292] Table 2 and Figure 3 The results showed that the PRLR chimeric antibody #885 could effectively bind to the PRLR antigen and had a greater binding ability than the chimeric antibody h16f disclosed in the existing patent, with an EC50 value that was 3.5 times higher (0.08724 / 0.02484=3.5). The negative control antibody HuIgG hardly bound to the PRLR antigen.

[0293] Example 7: In vitro tumor-inhibiting activity of PRLR chimeric antibody-drug conjugate.

[0294] After conjugating chimeric antibodies to toxic substances, cell killing experiments were used to verify whether chimeric antibodies could inhibit tumor activity in vitro.

[0295] 7.1 Preparation of PRLR chimeric antibody ch885 conjugated with the toxic substance MMAE

[0296] First, the disulfide bonds in the ch885 chimeric antibody structure were opened using the reducing agent TCEP (Thermo#20490). Then, the small molecule drug MMAE (VcMMAE, HY-15575 in this example) was added to the reduced antibody at an antibody:drug ratio of 1:14 for DAR8 conjugation. Unconjugated free small molecules were removed using a desalting column and activated carbon to obtain ADC-ch885-MMAE. Here, h16f-MMAE represents a chimeric antibody-drug conjugate disclosed in existing patents, and HuIgG-MMAE represents a negative control antibody-drug conjugate.

[0297] 7.1.1 Buffer Adjustment

[0298] The pH of the ch885 antibody was adjusted by adding prepared PBS (50mM phosphate + 2mM EDTA, pH=7.0).

[0299] 7.1.2 Reduction of disulfide bonds

[0300] Add antibody, TCEP, 5 / 10x PB and dimethyl water to a 1.5 mL centrifuge tube, mix well, and incubate at 37°C for 2 h for reduction.

[0301] 7.1.3 Conjugation of small molecule drugs

[0302] After reduction in a 37℃ constant temperature incubator, place it on ice to cool for 10 minutes.

[0303] First, add the DMA organic reagent (at room temperature) on ice, then add 10-15x of the small molecule drug and mix thoroughly.

[0304] Coupled by placing in a shaker at 22℃ (room temperature) for 1 hour.

[0305] 7.1.4 Desalination

[0306] The desalination column was equilibrated three times using 1x FB.

[0307] For formal experiments, samples are desalted twice. If the second sample is too small and falls below the desalting range of the desalting column, a small amount of FB can be added. Small-scale samples do not need to be desalted twice.

[0308] 7.1.5 Activated carbon adsorption of free small molecule drugs

[0309] Dissolve 300 mg of activated carbon in 1 mL of water / FB and add it to the antibody-drug conjugate system at a volume ratio of 10% (if adding powder directly, add 30 mg of activated carbon per 1 mL of sample).

[0310] Rotate at 22℃ or room temperature for 1–2 hours;

[0311] Centrifuge and aspirate the supernatant (subsequent experiments will be conducted in a clean bench).

[0312] 7.1.6 Filter Installation

[0313] The sample was filtered into a liquid chromatography vial containing a liner using a 0.22 μM filter on a clean bench.

[0314] HPLC was used for on-board detection, and HIC was used to detect the DAR value of the ADC. In the small-scale test, the gradient TCEP was used to conduct the experiment. The DAR and TCEP were curve fitted, and the amount of TCEP was calculated by the equation.

[0315] 7.1.7 Concentration Determination

[0316] The BCA method was used for detection.

[0317] 7.2 In vitro tumor inhibition test

[0318] Culture T47D (human breast duct carcinoma cells) and MDA-MB-231 (human breast cancer cells) cells, collect cells, seed them in 96-well plates, and adjust the cell density to 2–5 × 10⁻⁶ cells / well. 4 / mL. Soak 120μL per well, i.e., 5000 cells per well. Use the next day. Do not add cells to the outer ring of wells in the 96-well plate; add 200μL of sterile 1×PBS. Check that the incubator water bath is clean and has sufficient ddH2O. Incubate overnight. Primary antibody dilution: Prepare 10 concentrations (300000, 100000, 30000, 10000, 3000, 1000, 30, 100, 30, 10 pM) of chimeric antibody-drug conjugates, with 0 pM chimeric antibody-drug conjugate as a control. Use a 10-fold serial dilution of the antibody in a 96-well plate. Gently tap to mix, and continue incubation for 3-5 days, observing cell killing activity daily. CCK-8 assay. Dilute CCK-8 analytes 10-fold with RPMI-1640 (serum-free) for later use. Discard the culture medium from the 96-well plate and add 100 μL of diluted CCK8 to each well. Incubate at 37℃ for 0.5–2 h, and measure the absorbance at 450 nm. The OD value of the blank wells should be controlled to be around 1.5. Results are shown in Table 3. Figure 4 As shown:

[0319] Table 3

[0320]

[0321]

[0322] Table 3 and Figure 4The results showed that the PRLR chimeric antibody-drug conjugate ch-885-MMAE could inhibit tumor cell activity in vitro. Specifically, in the T47D cell killing results, low concentrations of ch-885-MMAE effectively killed T47D cells, achieving a killing effect of more than 20% at a concentration of 10 nM (10000 pM) and approximately 50% at a concentration of 100 nM (100000 pM). Furthermore, ch-885-MMAE at the same concentration had no significant killing effect on MDA-MB-231 cells.

[0323] The above results suggest that the antibodies and ADCs of the present invention can be endocytosed by binding to the PRLR receptor on the cell surface, thereby exerting the killing effect of ADC drugs in a targeted and efficient manner.

[0324] The sequence involved in this invention is shown below:

[0325] The VL sequence (SEQ ID NO:1) of antibody #885:

[0326] EIVLTQSPALMAASPGEKVTITTCSVSSSIRSTYLHWYQQKSGTSPKPWIYGTSNLASGVPVRFSGSGSGTSFSLTISSMEPEDAATYYCQQWSGFPLTIGAGTKLELK

[0327] #885 Antibody VLCDR1 Sequence (SEQ ID NO:2):SSIRSTY

[0328] #885 Antibody VLCDR2 Sequence (SEQ ID NO:3): GTS

[0329] #885 antibody VLCDR3 sequence (SEQ ID NO:4): QQWSGFPLT

[0330] VH sequence of antibody #885 (SEQ ID NO:5):

[0331] DVQLQESGPALIKPSQSLSLTCTVTGYSINSDYAWNWIRQFPGNKLEWMGYISYSGSTDYNPSLESRISITRDTSKNQFFLQLNSVTTEDTATYYCVRNNWYFDVWGAGTTVTVSS

[0332] #885 Antibody VHCDR1 Sequence (SEQ ID NO:6): GYSINSDYA

[0333] #885 Antibody VHCDR2 Sequence (SEQ ID NO:7): ISYSGST

[0334] VH CDR3 sequence of #885 antibody (SEQ ID NO:8): VRNNWYFDV

[0335] Heavy chain sequence of #885 antibody (SEQ ID NO:9):

[0336] DVQLQESGPALIKPSQSLSLTCTVTGYSINSDYAWNWIRQFPGNKLEWMGYISYSGSTDYNPSLESRISITRDTSKNQFFLQLNSVTTEDTATYYCVRNNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0337] Light chain sequence of #885 antibody (SEQ ID NO:10):

[0338] EIVLTQSPALMAASPGEKVTITCSVSSSIRSTYLHWYQQKSGTSPKPWIYGTSNLASGVPVRFSGSGSGTSFSLTISSMEPEDAATYYCQQWSGFPLTIGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0339] VH sequence of h16f patent antibody (SEQ ID NO:11):

[0340] EVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYWMHWVRQAPGQGLEWIGEIDPSDSYSNYNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARNGGLGPAWFSYWGQGTLVTVSS

[0341] VL sequence of the h16f patented antibody (SEQ ID NO:12):

[0342] DIQMTQSPSSVSASVGDRVTITCKASQYVGTAVAWYQQKPGKSPKLLIYSASNRYTGVPSRFSDSGSGTDFTLTISSLQPEDFATYFCQQYSSYPWTFGGGTKVEIK

[0343] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof targeting PRLR, characterized in that, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising the following complementarity determining regions (CDRs): The heavy chain variable region comprises the following complementarity determining regions (CDRs): a VHCDR1 as set forth in SEQ ID NO: 6, a VHCDR2 as set forth in SEQ ID NO: 7, and a VHCDR3 as set forth in SEQ ID NO: 8; and the light chain variable region comprises the following complementarity determining regions (CDRs): a VLCDR1 as set forth in SEQ ID NO: 2, a VLCDR2 as set forth in SEQ ID NO: 3, and a VLCDR3 as set forth in SEQ ID NO:

4.

2. A recombinant protein, characterized in that, The recombinant protein has: (i) the antibody or antigen-binding fragment thereof of claim 1 ; and (ii) optionally, a tag sequence to assist expression and / or purification.

3. A chimeric antigen receptor (CAR) comprising, The antigen binding domain of the chimeric antigen receptor contains a single chain variable region sequence scFv of an antibody targeting PRLR, the heavy chain variable region and the light chain variable region of the scFv comprising the following complementarity determining regions (CDRs): The heavy chain variable region comprises the following complementarity determining regions (CDRs): a VHCDR1 as set forth in SEQ ID NO: 6, a VHCDR2 as set forth in SEQ ID NO: 7, and a VHCDR3 as set forth in SEQ ID NO: 8; and the light chain variable region comprises the following complementarity determining regions (CDRs): a VLCDR1 as set forth in SEQ ID NO: 2, a VLCDR2 as set forth in SEQ ID NO: 3, and a VLCDR3 as set forth in SEQ ID NO:

4.

4. A polynucleotide, comprising: The polynucleotide encodes the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 2, or the chimeric antigen receptor CAR of claim 3.

5. A vector, characterized in that, The vector contains the polynucleotide of claim 4.

6. A host cell, characterized in that, The host cell contains the vector of claim 5 or the exogenous polynucleotide of claim 4 integrated in the chromosome.

7. A method of making a CAR-NK cell or a CAR-T cell, characterized in that, The CAR-NK cell or CAR-T cell expressing the chimeric antigen receptor of claim 3 comprises the following steps: transducing the polynucleotide of claim 4 or the vector of claim 5 into an NK cell or a T cell, thereby obtaining the CAR-NK cell or the CAR-T cell.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition contains the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 2, the chimeric antigen receptor of claim 3, the polynucleotide of claim 4, the vector of claim 5, or the host cell of claim 6, and a pharmaceutically acceptable carrier, diluent or excipient.

9. An immunoconjugate, the immunoconjugate comprising: (a) an antibody moiety selected from the group consisting of: the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 2, or a combination thereof; and (b) a conjugating moiety coupled to the antibody moiety, the conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

10. A method of producing a recombinant polypeptide, comprising, The method comprises: (a) culturing the host cell of claim 6 under conditions suitable for expression; (b) isolating from the culture the recombinant polypeptide, which is the antibody or antigen binding fragment thereof of claim 1, or the recombinant protein of claim 2.

Images