KR20190076324A - Anti-STAT3 bispecific antibody having cell penetrating ability - Google Patents

Abstract

The present invention relates to an anti-STAT3 bispecific antibody having a cell penetrating ability and, more specifically, to an anti-STAT3 bispecific antibody including a part specifically binding to STAT3 and a DNA binding part providing a cell penetrating ability. A STAT3/DNA bispecific antibody according to the present invention can overcome limitations of a conventional antibody medicine targeting only proteins existing outside a cell by including a DNA binding part penetrating into a cell and specifically binding to DNA in a nucleus, and can be effectively used for development of a therapeutic agent without side effects for related diseases caused by activation of STAT3 as it is confirmed that an antibody can specifically bind to only phosphorylated, activated STAT3 and inhibit a transcription factor activity thereof.

Description

STAT3 bispecific antibody having cell penetrating ability and anti-STAT3 bispecific antibody having cell penetrating ability

TECHNICAL FIELD The present invention relates to an anti-STAT3 bispecific antibody having intracellular ability to infiltrate, more specifically, to an anti-STAT3 bispecific antibody comprising a site specifically binding to STAT3 and a DNA binding site conferring intracellular infiltration ability .

Proteins are made from genetic information on DNA through transcription and translation. The protein is also a component of the cell and functions as a cytoskeleton. However, many proteins have an activity, and the regulation of the activity of the protein plays a very important role in the cell signaling for controlling the intracellular function.

Regulation of intracellular signal transduction by proteins can be achieved through on / off of protein activity. For induction of protein activation or inactivation, phosphorylation, glycosylation, methylation, acetylation acetylation, protein-protein interaction, and the like. Thus, post-translational modification plays a very important role in intracellular signal transduction. Of these, protein phosphorylation is a signal that is derived from the external and internal environment of the cell. (Manning G, et al., The protein kinase complement of the human genome. Science 298: 1912-1934 (2002)).

Such protein phosphorylation is known to play a role in intracellular signaling by influencing protein activity, structure, binding ability with other proteins, and the like. As molecular biology knowledge develops, new drugs based on these mechanisms are being developed. A representative example is a method of inhibiting the activity of a protein binding to the phosphorylated site of such a protein.

STAT3 (signal transducers and activators of transcription 3), a signal transduction protein that induces cell proliferation, induces phosphorylation by an extracellular signal such as interleukin (IL) or interferon-gamma (IFN-y) (Homo-dimer or hetero-dimer formation) into the nucleus and induce the expression of a specific gene. In particular, STAT3 has attracted much attention due to its known as a cancer inducing factor. In fact, abnormal activation of STAT3 has been reported in various cancer tissues, and accordingly, many researchers have participated in the development of an anticancer agent capable of inhibiting the abnormal activation of STAT3 . However, the development of drugs that can effectively inhibit STAT3 has not been developed yet.

Antibodies are glycoproteins present in serum or tissue fluids of all mammals and recognize foreign antigens in vivo. The antibody activates effector functions such as phagocytosis of FcR expressing cells, antibody-dependent cytotoxicity, free capacity of the mediator and antigen presenting ability through activation of the complement system and binding to a receptor (FcR) present on the cell surface, It is involved in biologic defense. Based on the antigen-specific binding ability of these antibodies, various drugs are being developed in the form of recombinant antibodies that bind to the substances responsible for intracellular diseases and inhibit their activity, and they are easier to produce than other types of drugs, , Specificity, and bio-durability.

However, a common antibody can only target extracellular molecules, and there are a number of important targets within the cell for the treatment of disease and the diagnosis of disease. For example, many transcription factors, particularly STAT3, are recognized as the most critical but challenging targets for the treatment of various diseases.

Accordingly, it is an object of the present invention to provide a method for solving the above-mentioned conventional limitations.

Disclosure of the Invention The present invention has been made in order to overcome the above-mentioned conventional limitations, and the present inventors have completed the present invention by developing a bispecific antibody that specifically binds to phosphorylated STAT3 and intracellular DNA and has cell penetration ability.

Accordingly, the present invention relates to a STAT3 / DNA bispecific antibody comprising a first antigen binding site specifically binding to STAT3 (signal transducer and activator of transcription 3) and a second antigen binding site specifically binding to DNA Or a fragment thereof.

It is another object of the present invention to provide a polynucleotide encoding said antibody or fragment thereof, a vector comprising said polynucleotide, and a cell transformed with said vector.

It is still another object of the present invention to provide a method for producing the antibody or fragment thereof and a STAT3-specific detection method.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention as described above, the present invention provides a method for detecting a first antigen binding site specifically binding to STAT3 (signal transducer and activator of transcription 3) and a second antigen binding site specifically binding to DNA A STAT3 / DNA bispecific antibody or fragment thereof.

In one embodiment of the present invention, the first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 1 to 3 and a light chain complementarity determining region And a heavy chain complementarity determining region represented by the amino acid sequence of SEQ ID NO:

In another embodiment of the present invention, the first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 7 to 9 and a light chain complementarity determining region And a heavy chain complementarity determining region represented by the amino acid sequence of SEQ ID NO:

In another embodiment of the present invention, the first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOs: 13 to 15 and a light chain complementarity determining region And a heavy chain complementarity determining region represented by the amino acid sequence of SEQ ID NO: 18.

In another embodiment of the present invention, the first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOs: 19 to 21 and a light chain complementarity determining region Chain complementarity determining region represented by the amino acid sequence of SEQ ID NO: 24.

In another embodiment of the present invention, the first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 25 to 27, Chain complementarity determining region represented by the amino acid sequence of SEQ ID NO: 30.

In another embodiment of the present invention, the first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 31 to 33, And a heavy chain complementarity determining region represented by the amino acid sequence of SEQ ID NO: 36.

In another embodiment of the present invention, the first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOs: 37 to 39, And a heavy chain complementarity determining region represented by the amino acid sequence of SEQ ID NO: 42.

In another embodiment of the present invention, the STAT3 may be a phosphorylated tyrosine residue at position 705.

In another embodiment of the present invention, the antibody or fragment thereof may be capable of penetrating into cells.

In another embodiment of the present invention, the fragment may be a fragment selected from the group consisting of diabodies, Fab, Fab ', F (ab) 2, F (ab') 2, Fv and scFv.

The present invention also provides a polynucleotide encoding said antibody or fragment thereof.

The present invention also provides a vector comprising the polynucleotide.

The present invention also provides a cell transformed with said vector.

(A) culturing the cells under conditions that express polynucleotides;

(b) producing a polypeptide comprising a light chain and a heavy chain variable region from said cell; And

(c) recovering the polypeptide or the STAT3 / DNA bispecific antibody or the fragment thereof from the cell or a culture medium in which the culture is cultured.

The present invention also provides a STAT3-specific detection method comprising contacting the antibody or fragment thereof with a sample and detecting the antibody or fragment thereof.

The STAT3 / DNA bispecific antibody according to the present invention can overcome limitations of an antibody drug targeting only proteins existing outside the cell by including a DNA binding site that penetrates into cells and specifically binds to DNA in the nucleus. , It could be specifically bound to the phosphorylated activated form of STAT3 alone and its transcription factor activity could be inhibited. As a result, it could be effectively used for the development of a therapeutic agent without side effects on the diseases caused by the activation of STAT3 will be.

FIG. 1A is a schematic view of the structure of a double antibody (hereinafter referred to as pSTAT3 antibody) specifically binding to phosphorylated STAT3, and FIG. 1B shows the antibody structure and its sequence of the present invention.
FIG. 2 shows the results of SDS-PAGE after the production of the pSTAT3 bispecific antibody of the present invention to confirm the binding ability to phosphorylated STAT3.
FIG. 3 shows the results of confirming the cell-penetrating ability of the pSTAT3 bispecific antibody of the present invention through immunocytochemical staining.
FIG. 4 is a graph showing the inhibitory effect of the pSTAT3 bispecific antibody of the present invention on pSTAT3. FIGS. 4A and 4C show the activity of STAT3 by treating LPS or TNFα in mouse macrophage cell line (RAW264.7) and joint bone marrow cells (Synoviocyte) FIG. 4C shows the expression level of pSTAT3 and COX-2 in the mouse macrophage cell line treated with LPS and pSTAT3, and the antibody of the present invention And the specific binding to pSTAT3 was confirmed.
FIG. 5 shows that the expression of the gene was inhibited by the treatment of the pSTAT3 bispecific antibody of the present invention under the condition that the luciferase reporter gene was expressed under the control of STAT3-RE when stimulated with IL-6 in HepG2 liver cancer cell line Results.

The present inventors have completed the present invention by developing bispecific antibodies that specifically bind to phosphorylated STAT3 and intracellular DNA and have cell-penetrating ability.

Accordingly, the present invention relates to a STAT3 / DNA bispecific antibody comprising a first antigen binding site specifically binding to STAT3 (signal transducer and activator of transcription 3) and a second antigen binding site specifically binding to DNA Or a fragment thereof.

The present inventors have produced STAT3 / DNA bispecific antibodies or fragments thereof by way of examples and verified their functions.

In one embodiment of the present invention, a total of 24 specific antibody sequences binding to the pY705 STAT3 peptide antigen were derived using a human scFv (single-chain variable fragment) library by the phage display method, and the affinity (KD) And 7 antibody candidates with high affinity for pY705 STAT3 were finally selected (see Example 1).

In another embodiment of the present invention, recombinant antibodies were prepared using three of the seven candidates, and SDA-PAGE was performed to confirm that pSTAT3 can be effectively detected by the antibody prepared using one of the two sequences (See Example 2).

In another embodiment of the present invention, the intracellular penetrating ability of the antibody was examined by a cell immunochemical staining method in a colon cancer cell line, and it was confirmed that the pSTAT3 antibody comprising the DNA binding domain had an effective intracellular permeability (Example 3).

In another embodiment of the present invention, the mouse macrophage cell line and joint bone marrow cells were treated with an antibody according to the present invention and the expression level of pSTAT3 was measured by Western blotting to confirm the pSTAT3 inhibitory activity of the antibody, (See Example 4-1).

In another embodiment of the present invention, it has been confirmed that the antibody inhibits the expression of the Luciferase reporter gene under IL-6 stimulation conditions using a luciferase reporter gene expressed under the control of STAT3-RE See Example 4-2).

The results of the above examples demonstrate that the STAT3 / DNA bispecific antibody according to the present invention has cell penetration ability and effectively inhibits the function of pSTAT3.

As used herein, the term " Antibody " includes immunoglobulin molecules which are immunologically reactive with specific antigens and include both polyclonal antibodies and monoclonal antibodies. The term also includes forms produced by genetic engineering such as chimeric antibodies (e. G., Humanized murine antibodies), heterologous binding antibodies (e. G., Bispecific antibodies), bispecific antibodies . In the present invention, the antibody is a bispecific antibody, which means an antibody in which one antibody has two binding sites specific to different antigens. The antibody of the present invention preferably binds to STAT3 and intracellular DNA, respectively .

STAT3 / DNA bispecific antibody 'and' STAT3 bispecific scFv antibody 'of the present invention are used most broadly in the present invention, and specific examples thereof are STAT3 Binding site that specifically binds to DNA and a binding site that specifically binds to DNA.

Typically, the antibody has a heavy chain and a light chain, and each heavy and light chain comprises a constant region and a variable region (also known as a " domain "). The variable regions of the light and heavy chains include three multifunctional regions called " complementarity-determining regions " (CDRs) and four framework regions. The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, CDR3 starting from the N-terminus and sequentially identified by the chain in which the particular CDR is located. However, not all CDR termini need to be directly involved in antigen binding.

In the present invention, the first antigen binding site specifically binding to STAT3 includes a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 1 to 3 and an amino acid sequence of SEQ ID NOS: Chain complementarity determining region represented by < RTI ID = 0.0 >

In addition, the first antigen binding site specifically binding to STAT3 includes a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 7 to 9 and an amino acid sequence of SEQ ID NO: And a heavy chain complementarity determining region.

In addition, the first antigen binding site specifically binding to STAT3 includes a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOs: 13 to 15 and a light chain complementarity determining region And a heavy chain complementarity determining region.

In addition, the first antigen binding site specifically binding to STAT3 has a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOs: 19 to 21 and an amino acid sequence represented by the amino acid sequence of SEQ ID NOs: And a heavy chain complementarity determining region.

In addition, the first antigen binding site specifically binding to STAT3 includes a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 25 to 27, and an amino acid sequence of SEQ ID NOS: And a heavy chain complementarity determining region.

Also, the first antigen binding site specifically binding to STAT3 includes a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 31 to 33 and an amino acid sequence of SEQ ID NOS: 34 to 36 And a heavy chain complementarity determining region.

The first antigen binding site specifically binding to STAT3 may be selected from the group consisting of a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOs: 37 to 39 and an amino acid sequence represented by the amino acid sequence of SEQ ID NOs: And a heavy chain complementarity determining region.

The fragment of the present invention may be a fragment selected from the group consisting of diabodies, Fab, Fab ', F (ab) 2, F (ab') 2, Fv and scFv, but is not limited thereto.

In the present invention, a fragment of an antibody refers to a fragment of an antibody that retains the antigen-specific binding force of the whole antibody. Preferably, the fragment has at least 20%, 50%, 70%, 80% %, 90%, 95% or 100% or more. Specifically, it may be in the form of Fab, F (ab) 2, Fab ', F (ab') 2, Fv, diabody, scFv and the like.

For purposes of the present invention, the fragment of the antibody is not limited in structure or form, but may preferably be scFv, as long as it retains the binding specificity for each of the human-derived STAT3 protein and DNA. The scFv according to the present invention has a CDR configuration specific to the above-mentioned STAT3 protein and DNA, or a VH and VL construct. When the C-terminus of VH and the N-terminus of VL are linked through a linker, Do not. The type of the linker is not particularly limited as long as it is known in the art as a linker applicable to scFv.

The antibody or fragment thereof of the present invention may comprise conservative amino acid substitutions (referred to as conservative variants of the antibody) that do not substantially alter its biological activity.

STAT3 is a transcription factor that is encoded by the STAT3 gene and is phosphorylated by receptor-associated Janus kinases (JAK) upon stimulation of various cytokines and growth factors to form a homodimer or heterodimer And it acts as a transcription factor that regulates the expression of various genes by moving to the nucleus. When interferon, epidermal growth factor (EGF), interleukin-5 (IL-5), or interleukin-6 (IL-6) binds to the ligand, phosphorylation of the 705th tyrosine residue of STAT3 It is known that phosphorylation occurs at the 727th serine residue by mitogen-activated protein kinases (MAPKs) or c-src non-receptor tyrosine kinases. Activated STAT3 mediates the expression of various genes in response to cell stimulation and plays an important role in various cell responses such as cell growth and apoptosis.

In the present invention, the STAT3 / DNA bispecific antibody or the fragment to which the STAT3 / DNA bispecific antibody binds is preferably STAT3 which is phosphorylated at the 705th tyrosine residue.

On the other hand, the second antigen binding site specifically binding to the DNA is a DNA binding domain of 3E10, which is an autoantibody found in lupus patients (J Autoimmun 1998 Oct; 11 (5): 539- 46. 1998.10), specifically, the 31st aspartic acid of the 'Variant heavy chain' is replaced with asparagine (D31N), but is not limited thereto.

The present invention also provides a polynucleotide encoding said antibody or fragment thereof.

The term " polynucleotide " used in the present invention may be described as an oligonucleotide or a nucleic acid, and may be a DNA molecule (e.g., cDNA or genomic DNA, RNA molecules (e.g., mRNA), a nucleotide analog (E. G., Peptide nucleic acids and non-naturally occurring nucleotide analogs) and hybrids thereof. The polynucleotides may be single-stranded ) Or double-stranded.

The polynucleotide of the present invention is not particularly limited in its sequence as long as it encodes the antibody of the present invention or a fragment thereof.

Polynucleotides encoding the antibodies or fragments thereof of the present invention can be obtained by methods well known in the art. For example, an oligonucleotide synthesis technique well known in the art, for example, a polymerase chain reaction (PCR), or the like may be used based on a DNA sequence or a corresponding amino acid sequence encoding a part or all of the heavy chain and light chain of the antibody . ≪ / RTI >

The present invention also provides a vector comprising the polynucleotide.

The term 'vector' used in the present invention is used for the purpose of cloning or expression of the polynucleotide of the present invention for recombinant production of the antibody or fragment thereof of the present invention. Generally, a vector, A marker gene, an enhancer element, a promoter, and a transcription termination sequence. The vector of the present invention may preferably be an expression vector, and more preferably, may be a vector comprising a polynucleotide of the present invention operably linked to a regulatory sequence, for example, a promoter.

The present invention also provides a cell transformed with said vector.

The cell of the present invention is not particularly limited as long as it is a cell that can be used to express an antibody or a polynucleotide encoding the fragment contained in the expression vector of the present invention. Cells (host cells) transformed with an expression vector according to the invention can be transformed into prokaryotic (e. G., E. coli), eukaryotes (e. G., Yeast or other fungi), plant cells (e. A mammalian cell, an animal cell (for example, a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, an insect cell or a hybridoma derived therefrom, May be cells derived from mammals, including humans.

The term " transformation " as used herein refers to a modification of the genotype of a host cell by the introduction of a foreign polynucleotide, and regardless of the method used for transformation, the foreign polynucleotide is introduced into the host cell . The exogenous polynucleotide introduced into the host cell may be maintained integrated or maintained in the genome of the host cell, but the present invention encompasses both.

The recombinant expression vector capable of expressing the STAT3 / DNA bispecific antibody or fragment thereof according to the present invention can be produced by a method known in the art, for example, transient transfection, microinjection, transduction, Cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, May be transformed into a cell for production of an antibody or a fragment thereof by electroporation, a gene gun, and a known method for introducing a nucleic acid into a cell, But is not limited to.

The present invention also relates to a method for producing a polypeptide comprising the steps of culturing the above cells under the conditions that the polynucleotide is expressed to produce a polypeptide comprising a light chain and a heavy chain variable region and recovering the polypeptide from the cell or a culture medium in which the polypeptide is cultured A STAT3 / DNA bispecific antibody, or a fragment thereof.

The culture of the cells may vary depending on the type of the cells, and may be appropriately selected and controlled by those skilled in the art.

The antibody molecule may be accumulated in the cytoplasm of a cell, secreted from the cell, targeted by a suitable signal sequence to a periplasm or extracellular medium (supernatant), and labeled with a periplasmic or extracellular medium . It is also desirable to refold the engineered antibody molecules using methods well known to those of ordinary skill in the art and to have functional conformation. The recovery of the polypeptide may vary depending on the characteristics of the produced polypeptide and the characteristics of the cells, and those skilled in the art can appropriately select and control the polypeptide.

The present invention also provides a STAT3-specific detection method comprising contacting the antibody or fragment thereof with a sample and detecting the antibody or fragment thereof.

A person skilled in the art can appropriately select a known method for detecting a protein using an antibody and prepare a sample suitable for a selected method. The sample may also be a cell or tissue, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, etc. obtained by biopsy or the like collected from a subject to be diagnosed as cancer or metastasis. The method for detecting a protein using the antibody is not limited to the above examples, and examples thereof include Western blot, immunoblot, dotslab, immunohistochemistry, enzyme immunoassay (ELISA), radioimmunoassay , Competitive binding assays, and immunoprecipitation.

The antibody or fragment thereof can generally be labeled with a detectable moiety for its detection. Examples of such enzymatic labels are luciferase such as Drosophila luciferase and bacterium luciferase, luciferin, luciferase such as luciferase, and luciferase such as luciferase, which may be labeled with, for example, radioactive isotopes or fluorescent labels, Peroxidase such as 2,3-dihydropthalazine dione, malate dehydrogenase, urase, horseradish peroxidase (HRPO), alkaline phosphatase,? -Galactosidase, (E. G., A free radical and a xanthine oxidase), a glucosamine oxidase, a glucosamine oxidase, and a glucose-6-phosphate dihydrogenase, Lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies can be conjugated directly or indirectly to the antibodies using known techniques. For example, the antibody can be conjugated to biotin and any label belonging to the three broad categories mentioned above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin, and thus this label can be conjugated to the antibody in this indirect manner.

 Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

EXAMPLES Example 1 Phosphorylated STAT3 Peptide Specific Antibody Sequence Detection and Candidate Selection Using Phagy Display

In order to prepare an antibody specifically binding to phosphorylated STAT3 (pY705 STAT3) at the 705th tyrosine residue, the present inventors first tried to screen the antibody binding to the phosphorylated STAT3 using a Phagy Display.

For this purpose, a human scFv (single-chain variable fragment) library provided from the Antibody Detection Support Team of the New Drug Development Support Center, Osong, Korea, was used and phage display was performed using pY705 STAT3 peptide as an antigen. Specifically, the immobilized antibody on the solid surface was mixed with a phage antibody library to induce antibody-antigen binding, and unbound phages were washed away. Next, the phage bound to the antigen is eluted using an alkaline / acidic pH solution or a competitor peptide, and then the E. coli is infected to amplify the eluted phage. And used for round panning to concentrate specific antibodies. As a result, 24 pY705 STAT3 peptide-specific antibody sequences were finally obtained.

As shown in the following Table 1, 7 antibody candidates having a KD of 90 nM or lower and having a high affinity for pY705 STAT3 were finally selected, and three candidate antibodies having a KD of 4-6 nM (C4 , 3A10, and C6) were selected for the development of recombinant double antibodies.

ID KD (nM) C4 4 3A10 5 C6 6 H9 12 A12 55 F11 55 A10 85

Example 2. Production of phosphorylated STAT3 specific recombinant antibodies

The present inventors made a recombinant double antibody specific for phosphorylated STAT3 (pY705 STAT3) using the three candidate antibody sequences selected in Example 1 above.

The double antibody produced in the present invention has a structure in which the antigen binding site at one end binds to phosphorylated STAT3 and the other antigen binding site binds to DNA in the nucleus of the cell as shown in Fig. More specifically, as shown in FIG. 1B, scFv (STAT3 scFv) binding to phosphorylated STAT3 and scFv (3E10 scFv) binding to DNA in the intracellular space penetrate into the nucleus are linked with a linker in the form of a swivel ). The 3E10 scFv is based on the DNA binding domain of an autoantibody found in lupus patients and is further characterized by the 31st aspartic acid of the 'Variant heavy chain' reported to be further enhanced in intracellular penetration efficiency ) Was replaced with asparagine (D31N).

Further, SDS-PAGE was performed to examine whether the phosphorylated STAT3 bispecific antibody (hereinafter, referred to as pSTAT3 antibody) produced by recombining with the above-described structure was expressed in cells and then bound to phosphorylated STAT3 Respectively. To this end, 100 μg of the recombinant pSTAT3 antibody-expressing plasmid DNA was introduced into CHO-K1 80M cells and expressed to obtain the corresponding antibody. Then, 19.5 μl of each sample was loaded on a 4-20% acrylamide gel and subjected to SDS-PAGE To examine whether the antibody targets and binds phosphorylated STAT3.

As a result, as shown in FIG. 2, it was confirmed that all of the pSTAT3 antibody (①-④) samples produced from cells through independent processes effectively bind to pSTAT3.

Example 3. Verification of cell penetration ability of pSTAT3 antibody

Cell permeability analysis was performed to verify whether the pSTAT3 antibody according to the present invention actually exhibits a function of penetrating into cells. For this, the pSTAT3 antibody (STAT3-D31N), which contains the pSTAT3 double antibody (pSTAT3-C4) containing 3E10 scFv or the 3E10 scFv and the 31st aspartic acid was replaced with asparagine (D31N) Next, STAT3 protein was labeled (STAT3 staining) by immunocytochemistry using each of the above pSTAT3 antibodies, and PI staining for nucleus was stained by PI staining.

As a result, as shown in FIG. 3, when the pSTAT3-D31N antibody was treated, the result of STAT3 staining was exactly the same as the result of PI staining the cell nucleus. As a result, it was confirmed that the antibody penetrates into cells and binds to pSTAT3 I have confirmed the ability. In contrast, the STAT3-C4 antibody also showed the ability to penetrate cells and bind to pSTAT3 in cells, but the cell penetration efficiency was lower than that of pSTAT3-D31N.

Example 4. Verification of pSTAT3 Inhibitory Effect of pSTAT3 Antibody

4-1. Verification of pSTAT3 inhibitory effect

From the results of Example 2, it was confirmed that the pSTAT3 antibody of the present invention binds efficiently to pSTAT3. Further, to examine the inhibitory effect of the antibody against pSTAT3, Western blotting was performed to observe the protein expression level.

More specifically, the mouse macrophage cell line RAW264.7 and the articular bone marrow cell Synoviocyte were treated with LPS (lipopolysaccharide) or TNFα to activate pSTAT3, and then the pSTAT3 antibody of the present invention was administered at 20, 50, 100 or 10, 20, 50 ug / ml) and the expression level of pSTAT3 protein was observed. As a result, as shown in Figs. 4A and 4B, when the pSTAT3 antibody (RSmu) was treated, the expression level of pSTAT3 was decreased in proportion to the treatment concentration in both cells. Thus, it can be seen that the pSTAT3 antibody according to the present invention effectively inhibits the pSTAT3 protein activated by LPS or TNF [alpha].

Furthermore, RAW264.7 cells were treated with LPS alone or with 50 ug of LPS and pSTAT3 antibody, and the level of COX-2 expression induced by NF-κB signaling was observed with pSTAT3. As a result, the expression of pSTAT3 protein was decreased while the expression level of COX-2 was not changed, as shown in FIG. 4C, confirming that the pSTAT3 antibody of the present invention did not affect the COX-2 protein. These results demonstrate that the antibody selectively inhibits the activated pSTAT3 protein and does not affect other signal transduction, thus proving that the side effects can be reduced when the antibody of the present invention is used.

4-2. Verification of STAT3-RE mediated gene expression inhibition

In addition to the results of Example 4-1, the present inventors examined whether the pSTAT3 antibody of the present invention can inhibit STAT-RE (Response element) mediated gene expression by inhibiting STAT3 function. For this, a luciferase reporter gene vector expressed under the control of STAT3-RE was transfected into HepG2 liver cancer cell line for 48 hours, and the pSTAT3 antibody of the present invention was treated for 1 hour, and 100 nM of IL- 6 was treated for 24 hours. Expression levels of the STAT3-RE mediated luciferase gene by IL-6 stimulation were then measured.

As a result, when the pSTAT3 antibody (D31N) was treated as shown in Fig. 5, it was confirmed that gene expression was almost completely inhibited as compared with the control (Cont). From the above results, it was found that the pSTAT3 antibody of the present invention has an effect of inhibiting the expression of STAT3-mediated gene.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> ROPHI BIO <120> Anti-STAT3 bispecific antibody having cell penetrating ability <130> PD17-188 <160> 42 <170> KoPatentin 3.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C4) _CDR1_Vk <400> 1 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn His Val Ser   1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C4) _CDR2_Vk <400> 2 Ser Asn Asn Gln Arg Pro Ser   1 5 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C4) _CDR3_Vk <400> 3 Ala Ser Trp Asp Tyr Ser Leu Asn Ala Tyr Val   1 5 10 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C4) _CDR1_VH <400> 4 Ser Tyr Tyr Met Ser   1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C4) _CDR2_VH <400> 5 Leu Ile Ser Pro Gly Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C4) _CDR3_VH <400> 6 Asp Leu Thr Ser Gln Leu Pro Asp Gly Phe Asp Tyr   1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (3A10) _CDR1_Vk <400> 7 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Ser   1 5 10 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (3A10) _CDR2_Vk <400> 8 Ser Asn Asn Gln Arg Pro Ser   1 5 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (3A10) _CDR3_Vk <400> 9 Gly Thr Trp Asp Tyr Ser Leu Ser Gly Tyr Val   1 5 10 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (3A10) _CDR1_VH <400> 10 Asn Tyr Ala Met Ser   1 5 <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (3A10) _CDR2_VH <400> 11 Gly Ile Ser His Asp Asp Gly Ser Lys Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (3A10) _CDR3_VH <400> 12 Gly Gly Ile Ser Cys Ser Arg Thr Gly Cys Tyr Ser Ala Asp Ala Met   1 5 10 15 Asp Val         <210> 13 <211> 13 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C6) _CDR1_Vk <400> 13 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Asn   1 5 10 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C6) _CDR2_Vk <400> 14 Tyr Asp Ser Lys Arg Pro Ser   1 5 <210> 15 <211> 11 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C6) _CDR3_Vk <400> 15 Gly Ser Trp Asp Ala Ser Leu Asn Gly Tyr Val   1 5 10 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C6) _CDR1_VH <400> 16 Asp Tyr Ala Met Ser   1 5 <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C6) _CDR2_VH <400> 17 Gly Ile Ser Ser Ser Gly Gly Ser Lys Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 18 <211> 18 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (C6) _CDR3_VH <400> 18 Phe Arg Arg Thr His Ser Thr Arg Asn Thr Ser Tyr Tyr Asn Gly Met   1 5 10 15 Asp Val         <210> 19 <211> 13 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (H9) _CDR1_Vk <400> 19 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Ser   1 5 10 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (H9) _CDR2_Vk <400> 20 Asp Asn Ser Gln Arg Pro Ser   1 5 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > pSTAT3 bispecific Ab (H9) _CDR3_Vk <400> 21 Ala Thr Trp Asp Asp Ser Leu Asn Gly Tyr Val   1 5 10 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (H9) _CDR1_VH <400> 22 Ser Tyr Asp Met Ser   1 5 <210> 23 <211> 17 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (H9) _CDR2_VH <400> 23 Val Ile Ser Pro Gly Ser Gly Ser Lys Tyr Tyr Ala Asp Ser Val Arg   1 5 10 15 Gly     <210> 24 <211> 7 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (H9) _CDR3_VH <400> 24 Ser Trp Gln Lys Phe Asp Tyr   1 5 <210> 25 <211> 13 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (A12) _CDR1_Vk <400> 25 Ser Arg Ser Ser Ser Asn Ile Gly Ser Asn Ser Val Thr   1 5 10 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > pSTAT3 bispecific Ab (A12) _CDR2_Vk <400> 26 Ser Asn Ser Gln Arg Pro Ser   1 5 <210> 27 <211> 11 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > pSTAT3 bispecific Ab (A12) _CDR3_Vk <400> 27 Ala Ser Trp Asp Asp Ser Leu Asn Gly Tyr Val   1 5 10 <210> 28 <211> 5 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (A12) _CDR1_VH <400> 28 Asn Tyr Ala Met Ser   1 5 <210> 29 <211> 17 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (A12) _CDR2_VH <400> 29 Ser Ile Ser Pro Gly Asn Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 30 <211> 18 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (A12) _CDR3_VH <400> 30 Ala Pro Arg His Cys Ser Met His Leu Cys Tyr Ser Ser Asn Ala Met   1 5 10 15 Asp Val         <210> 31 <211> 13 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (F11) _CDR1_Vk <400> 31 Thr Gly Ser Ser Ser Asn Ile Gly Asn Ser Ser Val Ser   1 5 10 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > pSTAT3 bispecific Ab (F11) _CDR2_Vk <400> 32 Ser Asp Ser Lys Arg Pro Ser   1 5 <210> 33 <211> 11 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > pSTAT3 bispecific Ab (F11) _CDR3_Vk <400> 33 Gly Thr Trp Asp Asp Ser Leu Ser Gly Tyr Val   1 5 10 <210> 34 <211> 5 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (F11) _CDR1_VH <400> 34 Gly Tyr Asp Met Ser   1 5 <210> 35 <211> 17 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (F11) _CDR2_VH <400> 35 Val Ile Ser Pro Gly Ser Gly Ser Lys Tyr Tyr Ala Asp Ser Val Arg   1 5 10 15 Gly     <210> 36 <211> 7 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (F11) _CDR3_VH <400> 36 Ser Phe Arg Thr Phe Asp Tyr   1 5 <210> 37 <211> 13 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > pSTAT3 bispecific Ab (A10) _CDR1_Vk <400> 37 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser   1 5 10 <210> 38 <211> 7 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > pSTAT3 bispecific Ab (A10) _CDR2_Vk <400> 38 Ala Asp Ser Lys Arg Pro Ser   1 5 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > pSTAT3 bispecific Ab (A10) _CDR3_Vk <400> 39 Gly Ala Trp Asp Ser Ser Leu Ser Gly Tyr Val   1 5 10 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (A10) _CDR1_VH <400> 40 Asp Tyr Ala Met Ser   1 5 <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <220> PSTAT3 bispecific Ab (A10) _CDR2_VH <400> 41 Trp Ile Ser Pro Gly Gly Ser Ser Ile Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > pSTAT3 bispecific Ab (A10) _CDR3_VH <400> 42 Gly Gly Arg Ala Tyr Arg Val Ala Phe Asp Tyr   1 5 10

Claims (16)

STAT3 / DNA bispecific antibody or fragment thereof comprising a first antigen binding site specifically binding to STAT3 (signal transducer and activator of transcription 3) and a second antigen binding site specifically binding to DNA.
The method according to claim 1,
The first antigen binding site specifically binding to STAT3 includes a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 1 to 3 and a heavy chain complementarity determining region Or a fragment thereof. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
The method according to claim 1,
The first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 7 to 9 and a heavy chain complementarity determining region represented by the amino acid sequence of SEQ ID NOS: Or a fragment thereof. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
The method according to claim 1,
The first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 13 to 15 and a heavy chain complementarity determining region represented by the amino acid sequence of SEQ ID NOs: 16 to 18 Or a fragment thereof. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
The method according to claim 1,
The first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOs: 19 to 21 and a heavy chain complementarity determining region represented by the amino acid sequence of SEQ ID NOs: Or a fragment thereof. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
The method according to claim 1,
The first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 25 to 27 and a heavy chain complementarity determining region Or a fragment thereof. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
The method according to claim 1,
The first antigen binding site specifically binding to STAT3 comprises a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOS: 31 to 33 and a heavy chain complementarity determining region represented by the amino acid sequence of SEQ ID NOS: 34 to 36 Or a fragment thereof. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
The method according to claim 1,
The first antigen binding site specifically binding to STAT3 includes a light chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOs: 37 to 39 and a heavy chain complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NOs: Or a fragment thereof. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
The method according to claim 1,
Wherein said STAT3 is a phosphorylated tyrosine residue at position 705, or a fragment thereof.
The method according to claim 1,
Wherein said antibody or fragment thereof is capable of penetrating into a cell.
The method according to claim 1,
Wherein the fragment is a fragment selected from the group consisting of diabodies, Fab, Fab ', F (ab) 2, F (ab') 2, Fv and scFv.
10. A polynucleotide encoding the antibody of claim 1 or a fragment thereof.
14. A vector comprising the polynucleotide of claim 12.
13. A cell transformed with the vector of claim 13.
(a) culturing the cells of claim 14 under conditions in which the polynucleotide is expressed;
(b) producing a polypeptide comprising a light chain and a heavy chain variable region from said cell; And
(c) recovering said polypeptide from said cell or a culture medium in which said cell has been cultured, or a method for producing said STAT3 / DNA bispecific antibody or fragment thereof.
Contacting the antibody or fragment thereof of claim 1 with a sample, and detecting the antibody or fragment thereof.

Images