Patent Application
20210363244
The present invention relates to an epitope of leucine-rich and immunoglobulin-like domains 1 (Lrig-1) protein, which is an antigen present on the surface of regulatory T cells, and an antibody or antigen-binding fragment specifically binding thereto.
One of the most important traits in all normal individuals is to have the ability to recognize and eliminate non-self antigens, while not detrimentally responding to antigenic substances that make up the self. As such, non-response of the living body to self antigens is called immunologic unresponsiveness or tolerance. Self-tolerance occurs by eliminating lymphocytes that may have specific receptors for self antigens, or by self-inactivation of the ability to respond after contacting self antigens. In a case where a problem arises in inducing or maintaining self-tolerance, an immune response to self antigens occurs, and the disease resulting therefrom is called autoimmune disease.
For the treatment of the autoimmune disease, a concept of suppressor T cells suggesting the possibility of presence of T cells capable of controlling and suppressing the effector function of conventional T cells was introduced and presented for the first time by Gershon in the early 1970s (R. K. Gershon and K. Kondo, Immunology, 1970, 18: 723-37). Since then, studies have been conducted to elucidate biological properties and functions of regulatory T cells in many areas of immunology.
In this connection, it has been reported that the regulatory T cells (Treg cells) play an important role in naturally preventing occurrence of excessive inflammation and immune responses; however, in a case where autoimmune disease and chronic inflammatory disease occur, the function and the number of the regulatory T cells are remarkably decreased. Therefore, in a case of patients with immune and inflammatory diseases, it is important that the regulatory T cells are produced at a normal level, which can be one of the treatments for these diseases.
Until now, studies on genes and proteins which are present specifically in regulatory T cells have been conducted, and it has been presented that substances such as CD25, CTLA4, CD62L, CD38, CD103, GITR, and CD45RB may correspond to marker substances. However, there are no genes and proteins that can target only the regulatory T cells alone.
On the other hand, there are three hypervariable regions called complementarity determining regions (hereinafter referred to as “CDRs”) and four framework regions. The CDRs primarily serve to bind to an epitope on an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 sequentially starting from the N-terminus, and are also distinguished by the chain where particular CDRs are located.
An object of the present invention is to provide an epitope of leucine-rich and immunoglobulin-like domains 1 (Lrig-1) protein present on the surface of regulatory T cells (Treg cells).
Another object of the present invention is to provide an antibody or antigen-binding fragment capable of specifically binding to the epitope.
Yet another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment capable of specifically binding to the epitope.
Still yet another object of the present invention is to provide a method for preventing or treating cancer, using the antibody or antigen-binding fragment capable of specifically binding to the epitope.
However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems which are not mentioned will be clearly understood by those skilled in the art from the following description.
The present invention relates to an epitope of leucine-rich and immunoglobulin-like domains 1 (Lrig-1) protein, or an antibody or antigen-binding fragment which specifically binds to the epitope.
In the present invention, the “Lrig-1 protein” is a transmembrane protein present on the surface of regulatory T cells, and is composed of leucine-rich repeats (LRRs) and three immunoglobulin-like domains on the extracellular or lumen side, a cell transmembrane sequence, and a cytoplasmic tail portion. The LRIG gene family includes LRIG1, LRIG2, and LRIG3, and the amino acids therebetween are highly conserved. The LRIG1 gene is highly expressed in normal skin and can be expressed in basal and hair follicle cells to regulate proliferation of epithelial stem cells. Therefore, the LRIG1 gene plays an important role in maintaining homeostasis of the epidermis, and its absence may develop psoriasis or skin cancer. It has been reported that in a case where chromosome 3p14.3 portion in which LRIG1 is located is cut off, there is a possibility of developing into cancer cells. In fact, it was identified that expression of LRIG1 is greatly decreased in renal cell carcinoma and cutaneous squamous cell carcinoma. Recently, it has been also found that Lrig-1 is expressed in only about 20 to 30% of cancers. On the other hand, for the purpose of the present invention, the Lrig-1 protein may be, but is not limited to, a protein present in humans or mice.
In an example of the present invention, the Lrig-1 protein may be Lrig-1 protein derived from mammals, including primates such as humans and monkeys, and rodents such as mice and rats.
In an example of the present invention, the Lrig-1 protein may be human-derived Lrig-1 protein represented by SEQ ID NO: 1, which may be encoded by, but is not limited to, a nucleic acid sequence represented by SEQ ID NO: 2 (see Table 1).
In another example of the present invention, the Lrig-1 protein may be, but is not limited to, mouse-derived Lrig-1 protein represented by SEQ ID NO: 3 (see Table 2).
According to an embodiment of the present invention, as the epitope of the Lrig-1 protein, there is provided an epitope including a polypeptide that consists of an amino acid sequence represented by Formula 1,
Lx1Lx2x3N. [Formula 1]
In Formula 1, x1 to x3 may each independently be a neutral amino acid, an acidic amino acid, a basic amino acid, or an aromatic amino acid. Here, the neutral amino acid may be glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), serine (S), or threonine (T); the acidic amino acid may be aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); the basic amino acid may be lysine (K), arginine (R), or histidine (H); and the aromatic amino acid may be phenylalanine (F) or tyrosine (Y).
In an example of the present invention, x1 to x3 may each independently be an amino acid selected from the group consisting of asparagine (N), aspartic acid (D), serine (S), tyrosine (Y), arginine (R), phenylalanine (F), lysine (K), histidine (H), leucine (L), valine (V), threonine (T), alanine (A), glutamine (Q), glutamic acid (E), and glycine (G).
In another example of the present invention, x1 may be an amino acid selected from the group consisting of asparagine (N), phenylalanine (F), aspartic acid (D), lysine (K), histidine (H), valine (V), arginine (R), and threonine (T); x2 may be an amino acid selected from the group consisting of serine (S), glutamine (Q), alanine (A), asparagine (N), glutamic acid (E), aspartic acid (D), phenylalanine (F), and glycine (G); and x3 may be an amino acid selected from the group consisting of tyrosine (Y), histidine (H), glycine (G), arginine (R), asparagine (N), leucine (L), lysine (K), and phenylalanine (F).
In yet another example of the present invention, x1 to x3 may each independently be an amino acid selected from the group consisting of asparagine (N), aspartic acid (D), serine (S), tyrosine (Y), and arginine (R).
In still yet another example of the present invention, x1 may be asparagine (N) or aspartic acid (D); x2 may be serine (S) or asparagine (N); and x3 may be tyrosine (Y) or arginine (R).
In the present invention, the epitope includes a polypeptide consisting of an amino acid sequence represented by Formula 1, and may consist of 10- to 20-mer, preferably 10- to 15-mer, and more preferably 11- to 14-mer.
In addition, in the present invention, the polypeptide consisting of the amino acid sequence represented by Formula 1 in the epitope may be located at positions 3 to 6, preferably positions 4 to 6, and more preferably position 5 from the N-terminus of the epitope.
As an example of the present invention, the polypeptide consisting of the amino acid sequence represented by Formula 1 may be represented by, but is not limited to, any one amino acid sequence of SEQ ID NOs: 4 to 17 in Table 3 below:
As another example of the present invention, the epitope may be represented by, but is not limited to, an amino acid sequence of SEQ ID NO: 18 or 20.
According to another embodiment of the present invention, as the epitope of the Lrig-1 protein, there is provided an epitope including a polypeptide represented by any one amino acid sequence of SEQ ID NOs: 18 to 29 in Table 4 below:
According to yet another embodiment of the present invention, there is provided a nucleic acid molecule encoding the epitope provided by the present invention.
The nucleic acid molecule of the present invention includes all nucleic acid molecules obtained by translating the amino acid sequences of the polypeptides provided by the present invention to polynucleotide sequences, as known to those skilled in the art. Therefore, various polynucleotide sequences may be prepared by an open reading frame (ORF), and all of these polynucleotide sequences are also included in the nucleic acid molecule of the present invention.
According to still yet another embodiment of the present invention, there is provided an expression vector into which the isolated nucleic acid molecule provided by the present invention is inserted.
In the present invention, the “vector” is a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a “plasmid,” which refers to circular double-stranded DNA into which an additional DNA segment can be ligated. Another type of vector is a phage vector. Yet another type of vector is a viral vector, where an additional DNA segment can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for example, bacterial vectors having a bacterial origin of replication are episomal mammalian vectors). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated along with the host genome. In addition, certain vectors are capable of directing expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” or simply “expression vectors.” In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
Specific examples of the expression vector in the present invention may be selected from, but are not limited to, the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμμ, T-even, T2, T3, T7; plant viruses. Any expression vector known, to those skilled in the art, as expression vectors can be used in the present invention, and the expression vector is selected depending on the nature of the target host cell. Introduction of a vector into a host cell may be performed by calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, lipofectamine transfection, or electroporation. However, the present invention is not limited thereto, and those skilled in the art may adopt and use an introduction method appropriate for the expression vector and the host cell which are used. The vector may preferably contain at least one selection marker. However, the present invention is not limited thereto, and selection can be made using the vector that contains no selection marker, depending on whether or not a product is produced. The selection marker is selected depending on the target host cell, which is done using methods already known to those skilled in the art, and thus the present invention has no limitation thereon.
In order to facilitate purification of the nucleic acid molecule of the present invention, a tag sequence may be inserted into and fused to an expression vector. The tag includes, but is not limited to, hexa-histidine tag, hemagglutinin tag, myc tag, or flag tag, and any tag known to those skilled in the art which facilitates purification can be used in the present invention.
According to still yet another embodiment of the present invention, there is provided a host cell line, transfected with the expression vector provided by the present invention.
In the present invention, the “host cell” includes individual cells or cell cultures which may be or have been recipients of the vector(s) for incorporation of a polypeptide insert. The host cell includes progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. The host cell includes cells transfected in vivo with the polynucleotide(s) herein.
In the present invention, the host cell may include cells of mammalian, plant, insect, fungal, or cellular origin, and may be, for example, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells such as yeast cells and Pichia pastoris; insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as CHO(Chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowes melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), or PERC.6 (human retinal cells); or plant cells. However, the host cell is not limited thereto, and any cell known to those skilled in the art which can be used as a host cell line is available.
According to still yet another embodiment of the present invention, there is provided an antibody or antigen-binding fragment which specifically binds to the epitope of the present invention.
In addition, the antibody according to the present invention specifically binds to an epitope including a polypeptide consisting of an amino acid sequence represented by Formula 1, or an epitope including a polypeptide represented by any one amino acid sequence of SEQ ID NOs: 18 to 29, in Lrig-1 protein present on regulatory T cells, so that the regulatory T cells' function can be suppressed and effector T cells' activity can be maintained or increased, thereby effectively suppressing growth of cancer cells, in particular, solid cancer cells.
As used herein, the “cancer” refers to or indicates a physiological condition characterized by cell growth in mammals which is not regulated in a typical manner. The cancer to be prevented, ameliorated, or treated in the present invention may be solid tumor formed of agglomerates caused by abnormal growth of cells in a solid organ, and may be, but is not limited to, gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, or the like, depending on location of the solid organ.
In the present invention, the antibody as a full-length antibody or as a part of the antibody has the ability to bind to the Lrig-1 protein, and includes any antibody fragment that binds to the Lrig-1 antigen determining site in a competitive manner with the binding molecule of the present invention.
As used herein, the “antibody” refers to a protein molecule which serves as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a particular antigen. For the purpose of the present invention, the antigen may be Lrig-1 protein present on the surface of regulatory T cells. Preferably, the antibody may specifically recognize the leucine-rich region or immunoglobulin-like domain of the Lrig-1 protein, but is not limited thereto.
In the present invention, the “immunoglobulin” has a heavy chain and a light chain, and each of the heavy chain and the light chain comprises a constant region and a variable region. The variable region of each of the light chain and the heavy chain contains three hypervariable regions called complementarity determining regions (hereinafter referred to as “CDRs”) and four framework regions. The CDRs primarily serve to bind to an epitope on an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 sequentially starting from the N-terminus, and are also distinguished by the chain where particular CDRs are located.
In the present invention, the “full-length antibody” has a structure with two full-length light chains and two full-length heavy chains in which each light chain is linked to a heavy chain by disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG. The IgG includes, as subtypes thereof, IgG1, IgG2, IgG3, and IgG4.
In addition, as used herein, the “antigen-binding fragment” refers to a fragment having an antigen-binding function, and examples of the antigen-binding fragment include (i) a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and a heavy chain constant region 1 (CH1); (ii) a Fd fragment consisting of VH and CH1 domains; (iii) a Fv fragment consisting of VL and VH domains of a single arm of an antibody; (iv) a dAb fragment (Ward, E. S. et al., Nature 341: 544-546 (1989)) consisting of a VH domain; (v) an isolated CDR region; (vi) a F(ab′)2 fragment, which is a bivalent fragment including two linked Fab fragments; (vii) a single-chain Fv molecule (scFv), in which a VH domain and a VL domain are linked by a peptide linker that allows the two domains to associate to form an antigen binding site; (viii) a bispecific single-chain Fv dimer; and (ix) a diabody, which is a multivalent or multispecific fragment constructed by gene fusion. The antigen-binding fragment may be obtained as a Fab or F(ab′)2 fragment in a case where a proteolytic enzyme, for example, papain or pepsin is used, and may be produced through a genetic recombinant technique.
In addition, in the present invention, the antibody may be, but is not limited to, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a bivalent, bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, or a tetrabody, or a fragment thereof.
In addition, as used herein, the “monoclonal antibody” refers to an antibody molecule of a single molecular composition which is obtained from substantially the same antibody population, and exhibits single binding specificity and affinity for a particular epitope.
In the present invention, the “chimeric antibody” is an antibody which is obtained by recombination of a variable region of a mouse antibody and a constant region of a human antibody, and has a greatly improved immune response as compared with the mouse antibody.
In addition, as used herein, the “humanized antibody” refers to an antibody obtained by modifying a protein sequence of an antibody derived from a non-human species so that the protein sequence is similar to an antibody variant naturally produced in humans. For example, the humanized antibody may be prepared as follows. Mouse-derived CDRs may be recombined with a human antibody-derived FR to prepare a humanized variable region, and the humanized variable region may be recombined with a constant region of a preferred human antibody to prepare a humanized antibody.
As used herein, the “binding” or “specific binding” refers to affinity of the antibody or antibody composition herein for an antigen. In antigen-antibody binding, the “specific binding” is distinguishable from non-specific background binding, typically in a case where a dissociation constant (Kd) is less than 1×10−5 M, less than 1×10−6 M, or less than 1×10−7 M. Specific binding can be detected by methods known in the art, such as ELISA, surface plasmon resonance (SPR), immunoprecipitation, and coprecipitation, which include an appropriate control that can distinguish between non-specific binding and specific binding.
The antibody or antigen-binding fragment of the present invention may exist as a multimer, such as a dimer, a trimer, a tetramer, or a pentamer, which includes at least part of antigen-binding capacity of a monomer. Such a multimer also includes a homomultimer or a heteromultimer. Antibody multimers contain a large number of antigen-binding sites, and thus have superior antigen-binding capacity as compared with monomers. Antibody multimers are also easily used to produce multifunctional (that is, bifunctional, trifunctional, tetrafunctional, or the like) antibodies.
As used herein, the “multifunctional” refers to an antibody or antigen-binding fragment which has two or more activities or functions (for example, antigen-binding capacity, enzyme activity, and ligand- or receptor-binding capacity). For example, the antibody of the present invention may be bound to a polypeptide having enzymatic activity, such as luciferase, acetyltransferase, and galactosidase, and the like. Multifunctional antibodies also include multivalent or multispecific (that is, bispecific, trispecific, or the like) forms of antibodies.
According to still yet another embodiment of the present invention, there is provided an antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment provided by the present invention and a drug.
As used herein, the “antibody-drug conjugate (ADC)” refers to a form in which the drug and the antibody are chemically linked to each other without degrading biological activity of the antibody and the drug. In the present invention, the antibody-drug conjugate denotes a form in which the drug is bound to an amino acid residue at the N-terminus of the heavy and/or light chain of the antibody, specifically, a form in which the drug is bound to an α-amine group at the N-terminus of the heavy and/or light chain of the antibody.
As used herein, the “drug” may mean any substance having a certain biological activity for a cell, which is a concept including DNA, RNA, or a peptide. The drug may be in a form which contains a reactive group capable of reacting and crosslinking with an α-amine group, and also includes a form which contains a reactive group capable of reacting and crosslinking with an α-amine group and to which a linker is linked.
In the present invention, examples of the reactive group capable of reacting and crosslinking with the α-amine group are not particularly limited in terms of type as long as the reactive group can react and crosslink with an α-amine group at the N-terminus of a heavy or light chain of an antibody. The reactive group includes all types of groups known in the art which react with an amine group. The reactive group may, for example, be any one of isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester, but is not limited thereto.
In the present invention, the antibody-drug conjugate includes, as the antibody or antigen-binding fragment, an antibody or antigen-binding fragment which specifically binds to the epitope of the present invention, that is, an epitope including a polypeptide consisting of an amino acid sequence represented by Formula 1, or an epitope including a polypeptide represented by any one amino acid sequence of SEQ ID NOs: 18 to 29, in Lrig-1 protein, in which the drug may be a drug that can treat cancer, a disease targeted by a Lrig-1 antibody, that is, an anticancer agent.
In the present invention, the anticancer agent may include any drug without limitation as long as the drug is used for prevention, amelioration, or treatment of cancer. The anticancer agent may be, for example, selected from the group consisting of nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semaxanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, Viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tiuxetan, heptaplatin, methyl aminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracil, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludarabine, enocitabine, flutamide, capecitabine, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vinblastine, idarubicin, mitomycin, bleomycin, dactinomycin, pirarubicin, aclarubicin, peplomycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretinoin, exemestane, aminogluthetimide, anagrelide, olaparib, navelbine, fadrozole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, 5FU, vorinostat, entinostat, and carmustine. However, the anticancer agent is not limited thereto.
In the present invention, the cancer may be solid tumor formed of agglomerates caused by abnormal growth of cells in a solid organ, and specific examples thereof may include, but are not limited to, gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, or the like, depending on location of the solid organ.
According to still yet another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, the antibody or antigen-binding fragment provided by the present invention, or the antibody-drug conjugate (ADC) provided by the present invention.
In the present invention, the antibody or antigen-binding fragment, or the antibody-drug conjugate obtained by binding a drug thereto, which is contained as an active ingredient in the pharmaceutical composition, specifically binds to an epitope including a polypeptide consisting of an amino acid sequence represented by Formula 1, or an epitope including a polypeptide represented by any one amino acid sequence of SEQ ID NOs: 18 to 29, in Lrig-1 protein present on regulatory T cells, so that the regulatory T cells' function can be suppressed and effector T cells' activity can be maintained or increased, thereby effectively suppressing growth of cancer cells, in particular, solid cancer cells.
In the present invention, the cancer may be solid tumor formed of agglomerates caused by abnormal growth of cells in a solid organ, and specific examples thereof may include, but are not limited to, gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, or the like, depending on location of the solid organ.
Meanwhile, in the present invention, the “prevention” may include, without limitation, any act of blocking symptoms of a disease, or suppressing or delaying the symptoms, using the pharmaceutical composition of the present invention.
In addition, in the present invention, the “treatment” may include, without limitation, any act of ameliorating or beneficially altering symptoms of a disease, using the pharmaceutical composition of the present invention.
In the present invention, the pharmaceutical composition may be characterized by being in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and the pharmaceutical composition may be characterized by being targeted to humans.
In the present invention, the pharmaceutical composition may be formulated in the form of oral preparations such as powders, granules, capsules, tablets, and aqueous suspensions, preparations for external use, suppositories, and sterile injectable solutions, respectively, according to conventional methods, and used. However, the pharmaceutical composition is not limited thereto. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, a binder, a glidant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a flavor, and the like may be used for oral administration; a buffer, a preserving agent, a pain-relieving agent, a solubilizer, an isotonic agent, a stabilizer, and the like may be used in admixture for injections; and a base, an excipient, a lubricant, a preserving agent, and the like may be used for topical administration. The preparations of the pharmaceutical composition of the present invention may be prepared in various ways by being mixed with the pharmaceutically acceptable carrier as described above. For example, for oral administration, the pharmaceutical composition may be formulated in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, or the like. For injections, the pharmaceutical composition may be formulated in the form of unit dosage ampoules or multiple dosage forms. Alternatively, the pharmaceutical composition may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, or the like.
Meanwhile, as examples of carriers, diluents, or excipients suitable for making preparations, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, or the like may be used. In addition, a filler, an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, and the like may further be included.
The route of administration of the pharmaceutical composition of the present invention includes, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal route. Oral or parenteral administration is preferred.
In the present invention, the “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrabursal, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of suppositories for rectal administration.
The pharmaceutical composition of the present invention may vary depending on a variety of factors, including activity of a certain compound used, the patient's age, body weight, general health status, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and severity of a certain disease to be prevented or treated. A dose of the pharmaceutical composition may vary depending on the patient's condition, body weight, severity of disease, drug form, route of administration, and duration, and may be appropriately selected by those skilled in the art. The pharmaceutical composition may be administered in an amount of 0.0001 to 50 mg/kg or 0.001 to 50 mg/kg, per day. Administration may be made once a day or several times a day. The dose is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated in the form of pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.
According to still yet another embodiment of the present invention, there is provided a method for preventing or treating cancer, comprising a step of administering, to an individual, the antibody or antigen-binding fragment according to the present invention, or the antibody-drug conjugate (ADC) according to the present invention.
The antibody or antigen-binding fragment of the present invention, and the antibody-drug conjugate of the present invention specifically bind to an epitope including a polypeptide consisting of an amino acid sequence represented by Formula 1, or an epitope including a polypeptide represented by any one amino acid sequence of SEQ ID NOs: 18 to 29, in Lrig-1 protein present on regulatory T cells, so that the regulatory T cells' function can be suppressed and effector T cells' activity can be maintained or increased, thereby effectively suppressing growth of cancer cells, in particular, solid cancer cells.
In the present invention, the “individual” is an individual suspected of developing cancer, and the individual suspected of developing cancer means a mammal, such as humans, mice, and domestic animals, who has developed or is likely to develop the disease in question. However, any individual, who is treatable with the antibody or antibody-drug conjugate of the present invention, is included therein without limitation.
The method of the present invention may comprise administering the antibody or the antibody-drug conjugate in a pharmaceutically effective amount. An appropriate total daily amount used may be determined by an attending physician or veterinarian within the scope of sound medical judgment, and administration may be made once or several times. However, for the purposes of the present invention, a specific therapeutically effective amount for a particular patient is preferably applied differently depending on various factors, including type and degree of reaction to be achieved, the specific composition including whether other agents are used therewith as the case may be, the patient's age, body weight, general health status, sex, and diet, time of administration, route of administration, secretion rate of the composition, duration of treatment, and drugs used simultaneously or in combination with the specific composition, and similar factors well known in the medical field.
Meanwhile, the method for preventing or treating cancer may be, but is not limited to, a combination therapy that further comprises administering a compound or substance having therapeutic activity against one or more cancer diseases.
In the present invention, the “combination” should be understood to represent simultaneous, individual, or sequential administration. In a case where the administration is made in a sequential or individual manner, the second component should be administered at intervals such that beneficial effects of the combination are not lost.
In the present invention, a dosage of the antibody or antibody-drug conjugate may be, but is not limited to, about 0.0001 μg to 500 mg per kg of patient's body weight.
In the present invention, the cancer may be solid tumor formed of agglomerates caused by abnormal growth of cells in a solid organ, and specific examples thereof may include, but are not limited to, gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, or the like, depending on location of the solid organ.
The antibody or antigen-binding fragment which specifically binds to an epitope of Lrig-1 protein, according to the present invention, specifically binds to the epitope of the present invention present on regulatory T cells, so that the regulatory T cells' function can be suppressed and effector T cells' activity can be maintained or increased, thereby effectively suppressing growth of cancer cells, in particular, solid cancer cells.
The present invention relates to an epitope of leucine-rich and immunoglobulin-like domains 1 (Lrig-1) protein, or an antibody or antigen-binding fragment which specifically binds to the epitope.
According to an embodiment of the present invention, as the epitope of the Lrig-1 protein, there is provided an epitope including a polypeptide that consists of an amino acid sequence represented by Formula 1,
Lx1Lx2x3N. [Formula 1]
In Formula 1, x1 to x3 may each independently be a neutral amino acid, an acidic amino acid, a basic amino acid, or an aromatic amino acid. Here, the neutral amino acid may be glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), serine (S), or threonine (T); the acidic amino acid may be aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q); the basic amino acid may be lysine (K), arginine (R), or histidine (H); and the aromatic amino acid may be phenylalanine (F) or tyrosine (Y).
In an example of the present invention, x1 to x3 may each independently be an amino acid selected from the group consisting of asparagine (N), aspartic acid (D), serine (S), tyrosine (Y), arginine (R), phenylalanine (F), lysine (K), histidine (H), leucine (L), valine (V), threonine (T), alanine (A), glutamine (Q), glutamic acid (E), and glycine (G).
In another example of the present invention, x1 may be an amino acid selected from the group consisting of asparagine (N), phenylalanine (F), aspartic acid (D), lysine (K), histidine (H), valine (V), arginine (R), and threonine (T); x2 may be an amino acid selected from the group consisting of serine (S), glutamine (Q), alanine (A), asparagine (N), glutamic acid (E), aspartic acid (D), phenylalanine (F), and glycine (G); and x3 may be an amino acid selected from the group consisting of tyrosine (Y), histidine (H), glycine (G), arginine (R), asparagine (N), leucine (L), lysine (K), and phenylalanine (F).
In yet another example of the present invention, x1 to x3 may each independently be an amino acid selected from the group consisting of asparagine (N), aspartic acid (D), serine (S), tyrosine (Y), and arginine (R).
In still yet another example of the present invention, x1 may be asparagine (N) or aspartic acid (D); x2 may be serine (S) or asparagine (N); and x3 may be tyrosine (Y) or arginine (R).
In still yet another example of the present invention, the polypeptide consisting of the amino acid sequence represented by Formula 1 may be represented by, but is not limited to, any one amino acid sequence of SEQ ID NOs: 4 to 17.
According to another embodiment of the present invention, as the epitope of the Lrig-1 protein, there is provided an epitope including a polypeptide represented by any one amino acid sequence of SEQ ID NOs: 18 to 29 in Table 4.
According to yet another embodiment of the present invention, there is provided an antibody or antigen-binding fragment which specifically binds to the epitope of the present invention.
According to still yet another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, the antibody or antigen-binding fragment provided by the present invention.
Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for describing the present invention in more detail, and it will be apparent to those skilled in the art that according to the gist of the present invention, the scope of the present invention is not limited by these examples.
In order to identify whether the Lrig-1 protein is expressed only in regulatory T cells (Treg) cells, the subsets of T cells, Th0, Th1, Th2, Th17, and iTreg, were prepared. The iTreg refers to cells whose differentiation has been artificially induced in a medium having the following composition, unlike nTreg which has been naturally isolated.
The subsets of the T cells were induced to differentiate into respective cells by first isolating naive T cells obtained from the spleen of mice, causing RPMI1640 (Invitrogen Gibco, Grand Island, N.Y.) nutrient medium that contains 10% fetal bovine serum (FBS; HyClone, Logan, Utah) to further contain the respective ingredients of Table 5 below, and performing 72-hour incubation in an incubator at 37° C., 5% CO2.
A three-dimensional steric structure of the extracellular domain of the Lrig-1 protein was predicted to produce an antibody specific for the Lrig-1 protein, a surface protein of regulatory T cells.
First, in order to predict base sequences of epitopes, tools of Uniprot (http://www.uniprot.org) and RCSB Protein Data Bank (http://www.rcsb.org/pdb) were used to predict a three-dimensional steric structure of the extracellular domain (ECD) of the Lrig-1 protein so that the structure of ECD is identified. Then, the results are illustrated in
As illustrated in
In addition, as illustrated in
Verification was made of whether the Lrig-1 protein can act as a biomarker specific for regulatory T cells.
For the verification, CD4+ T cells were isolated using magnet-activated cell sorting (MACS), through CD4 beads, from the spleen of mice. Subsequently, regulatory T (CD4+ CD25+ T) cells and non-regulatory T (CD4+CD25− T) cells were isolated with a fluorescence-activated cell sorter (FACS) using a CD25 antibody. For the respective cells and the cells differentiated in Preparation Example 1, mRNA was extracted using Trizol, and then gDNA was removed from genomic RNA using gDNA extraction kit (Qiagen) according to the protocol provided by the manufacturer. The gDNA-removed mRNA was synthesized into cDNA through the BDsprint cDNA Synthesis Kit (Clonetech).
Real-time polymerase chain reaction (RT PCR) was performed to quantitatively identify an expression level of Lrig-1 mRNA in the cDNA.
The real-time polymerase chain reaction was performed with primers shown in Table 6 below using SYBR Green (Molecular Probes) according to the protocol provided by the manufacturer under conditions of 40 cycles consisting of 95° C. for 3 minutes, 61° C. for 15 seconds, 72° C. for 30 seconds; and a relative gene expression level was calculated using the ΔCT method, and normalized using HPRT. The results are illustrated in
As illustrated in
In addition, as illustrated in
From the above results, it can be seen that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells, in particular, naturally-occurring regulatory T cells.
It was identified whether the Lrig-1 protein expressed from Lrig-1 mRNA is specifically expressed only in regulatory T cells.
Using FOXP3-RFP-knocked-in mice, the FOXP3-RFP obtained by coupling red fluorescence protein (RFP) to FOXP3 promoter, which is a transcription factor specific for regulatory T cells, CD4+ T cells were isolated using magnet-activated cell sorting (MACS), through CD4 beads, from the spleen of the mice. Subsequently, using RFP protein, regulatory T (CD4+RFP+ T) cells and non-regulatory T (CD4+RFP− T) cells were obtained by performing isolation through a fluorescence-activated cell sorter (FACS). The respective cells were stained with the purchased Lrig-1 antibody and a negative control was stained with an isotype-matched control antibody, to measure an expression level of Lrig-1 with the fluorescence-activated cell sorter. The results are illustrated in
As illustrated in
From the above results, it can be seen that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells.
From the viewpoint that in order to be a target of cell therapy, the Lrig-1 protein must be expressed on the surface of regulatory T cells, which in turn allows a more effective target therapy, it was identified whether the Lrig-1 protein is expressed on the surface of the regulatory T cells.
The respective differentiated T cell subsets of Preparation Example 1 were stained with anti-CD4-APC and anti-Lrig-1-PE antibodies, and expression levels of Lrig-1 were measured at the respective cell surfaces using a fluorescence-activated cell sorter (FACS). The results are illustrated in
As illustrated in
From the above results, it can be seen that the Lrig-1 protein according to the present invention is not only specifically expressed in regulatory T (Treg) cells, but also is, in particular, expressed at a higher level on the surface of the Treg cells.
Antibodies specific for the Lrig-1 protein according to the present invention were produced. The present antibodies were not produced by specifying a certain epitope, but were produced as antibodies capable of binding to any site on the Lrig-1 protein.
In order to produce the antibodies, cells expressing the Lrig-1 protein were produced. More specifically, a DNA fragment corresponding to SEQ ID NO: 2 and pcDNA (hygro) were cleaved with a cleavage enzyme, incubated at 37° C., and ligated to produce pcDNA into which a DNA sequence of the Lrig-1 protein is inserted. The thus produced pcDNA into which SEQ ID NO: 2 is inserted was introduced, through transfection, into L cells, so that the Lrig-1 protein is allowed to be expressed on the surface of the L cells.
Light and heavy chain amino acid sequences capable of binding to Lrig-1 expressed on the cell surface were selected from the Human scFv library so that a total of eight heavy and light chains were selected.
The selected heavy and light chain amino acid sequences were fused with the mlgG2a Fc region, to produce monoclonal antibodies. The sequences of the monoclonal antibodies are shown in Table 7 below.
In order to identify whether the monoclonal antibodies according to the present invention produced in Production Examples 1 to 8 well recognize Lrig-1, each of the antibodies of Production Examples 1 to 8 was bound to L cells that stably express Lrig-1. Then, a secondary antibody which is conjugated with eFlour 670 and is capable of recognizing mouse antibodies was added thereto, and then binding capacity of the monoclonal antibodies to the Lrig-1 protein was analyzed using FACS. The results are illustrated in
As illustrated in
In order to analyze how the monoclonal antibodies according to the present invention produced in Production Examples 1 to 8 affect the signal transduction pathway in regulatory T cells through the Lrig-1 protein, Lrig-1 present on the surface of the regulatory T cells was stimulated by treating the regulatory T cells with the antibodies of Production Examples 1 to 8, and then a level of tyrosine phosphorylation of Stat3 protein present in the stimulated regulatory T cells was analyzed through phosphotyrosine immunoblot. The results are illustrated in
As illustrated in
In order to identify therapeutic effects, on solid cancer, of the monoclonal antibodies (A8, B8, D9, and H6) according to the present invention, which had been produced in Production Examples 5 to 8, as illustrated in
As illustrated in
From these results, it can be seen that the Lrig-1 protein-specific monoclonal antibodies according to the present invention suppress growth of various solid cancer cells, thereby effectively preventing, ameliorating, or treating such cancers.
In order to discover epitopes that bind to the H6 antibody of Production Example 8, in which the epitopes are present in respective LRR domains and Ig-like domains in the Lrig-1 protein present on the surface of regulatory T cells, the following experiment was performed.
1. Experimental Preparation
The Lrig-1 protein was elongated by GSGSGSG linkers at the C- and N-termini thereof to avoid truncation of peptides. The elongated antigenic sequence was translated into 15 amino acids with a peptide-peptide overlap of 14 amino acids. The resulting Lrig-1 peptide microarrays contained 1,091 different peptides printed in duplicates and framed by additional HA (YPYDVPDYAG, 102 spots) control peptides.
(2) Experimental Material
Sample: H6 monoclonal antibody of Production Example 8
Washing buffer: PBS, pH 7.4 with 0.05% Tween 20 (3 times, 10 seconds after each incubation)
Blocking buffer: Rockland blocking buffer MB-070 (30 minutes before first assay)
Incubation buffer: wash buffer with 10% blocking buffer
Assay conditions: antibody concentration in incubation buffer being adjusted to 1 μg/ml, 10 μg/ml, and 100 μg/ml; and incubation being performed at 4° C. for 16 hours, and stirring being performed at 140 rpm
Secondary antibody: goat anti-human IgG (H+L) DyLight680 (0.2 μg/ml); staining being performed in incubation buffer at room temperature for 45 minutes
Control antibody: mouse monoclonal anti-HA (12CA5) DyLight800 (0.5 μg/ml); staining being performed in incubation buffer at room temperature for 45 minutes
Scanner: LI-COR Odyssey Imaging System; scanning offset 0.65 mm, resolution 21 scanning intensity of 7/7 (red=700 nm/green=800 nm)
2. Experimental Method
Pre-staining of a Lrig-1 peptide microarray copy using secondary and control antibodies in incubation buffer was performed to identify whether they can interact with antigen-derived peptides that can interfere with the main assays. Subsequent incubation of other Lrig-1 peptide microarray copies with the H6 monoclonal antibody of Production Example 8 (1 μg/ml, 10 μg/ml, and 100 μg/ml) in incubation buffer was followed by staining with secondary and control antibodies. Read-out was performed at a scanning intensity of 7/7 (red/green) using the LI-COR Odyssey Imaging System. Quantification of spot intensities and peptide annotation were based on the 16-bit gray scale tiff files. Microarray image analysis was done with PepSlide® Analyzer. The results are illustrated in
3. Experimental Results
As illustrated in
Although the present invention has been described in detail above, the scope of the present invention is not limited thereto. It will be obvious to those skilled in the art that various modifications and changes can be made without departing from the technical spirit of the present invention described in the claims.
The present invention relates to an epitope of leucine-rich and immunoglobulin-like domains 1 (Lrig-1) protein, which is an antigen present on the surface of regulatory T cells, and an antibody or antigen-binding fragment specifically binding thereto.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0053052 | May 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/005592 | 5/9/2019 | WO | 00 |
