KR20230103244A - Double-targeting protein comprising anti-HER2 scFv and peptide for targeting CP2c and use thereof - Google Patents

Abstract

The present invention relates to dual target proteins comprising anti-HER2 scFv and CP2c target peptides and uses thereof.
The dual target protein according to the present invention includes an anti-HER2 scFv and a CP2c targeting peptide, so that HER2 targeting by the anti-HER2 scFv and CP2c targeting by the peptide can be simultaneously expressed. Accordingly, the dual target protein of the present invention can improve the low complete remission rate seen in conventional anti-HER2 antibody-based anticancer drugs while maintaining targeting to HER2.
In addition, the dual target protein of the present invention secures stability that can last for a long time in vivo, can selectively remove only various types of cancer cells, and can act on drug-resistant cancer cells.

Description

Double-targeting protein comprising anti-HER2 scFv and peptide for targeting CP2c and use thereof

The present invention relates to dual target proteins comprising anti-HER2 scFv and CP2c target peptides and uses thereof.

Antibody therapy has been established for the targeted treatment of patients with cancer, immune disorders and angiogenic disorders [Carter, P., (2006) Nature Reviews Immunology 6:343-357]. Antibody-drug conjugates (ADCs) for local delivery of cytotoxic agents or cytostatic agents are immunoconjugates that are used to kill or inhibit tumor cells in the treatment of cancer. ) and aims to accumulate it in tumor cells [Junutula, et al., 2008b Nature Biotech. 26(8):925-932; Dornan, et al (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; WO2009/052249; McDonagh, (2006) Protein Eng. Design & Sel. 19(7): 299-307; Doronina, et al., (2006) Bioconj. Chem. 17:114-124; Erickson, et al., (2006) Cancer Res. 66(8):1-8; Sanderson, et al., (2005) Clin. Cancer Res. 11:843-852; Jeffrey, et al., (2005) J. Med. Chem. 48:1344-1358; Hamblett, et al., (2004) Clin. Cancer Res. 10:7063-7070].

Among the human epidermal growth factor receptor (HER/EGFR/ERBB) family, Human EGFR2 (HER2) is known as HER2/neu or ErbB2 as a site that receives extracellular signals, and as a site that conducts intracellular signal transmission, tyrosine kinase receptor kinase receptors). It shows a very high structural similarity rate with the other three EGFR families (HER1, HER3, HER4). Although the ligand that sends a signal that binds to HER2 is not yet known, it forms homodimers or heterodimers in partnership with other HER receptors that bind to ligands, increasing cell cycle, regulating cell proliferation, and It is known to be involved in differentiation and survival.

For the treatment of HER2-positive breast cancer, Perjeta (Pertuzumab), a HER2 dimerization inhibitor, Herceptin (Trastuzumab), a HER2-targeting anticancer drug, and Kesylar, a second-line treatment for HER2-positive metastatic breast cancer [Therapeutics such as Kadcyla (trastuzumab emtansine, Trastuzumab emtansine) and capecitabine are used in combination with lapatinib, which only acts on HER2-positive breast cancer, and has been approved for the treatment of HER2-positive breast cancer. accepted and used. In addition, not only breast cancer, but also colorectal cancer, gallbladder cancer, cholangiocarcinoma, bladder cancer, ovarian cancer, uterine cancer, pancreatic cancer, prostate cancer, brain cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer , As overexpression of the HER2 gene has been observed in esophageal cancer and gastric cancer, studies on the use of HER2-related anticancer drugs in combination or related treatments are currently underway in lung cancer and gastric cancer.

As a HER2-positive anticancer drug, Herceptin (trastuzumab) is a humanized monoclonal antibody for recombinant humanization therapy that targets the extracellular domain of the HER2 protein. Herceptin (trastuzumab) is effective only when the gene protein HER2 receptor is positive. It has been reported that recurrence rate is reduced by 50% and mortality rate by 30% when administered for 1 year in not only recurrent/hypermetastatic breast cancer but also in early breast cancer. It is evaluated as an important drug in the development of therapeutic agents.

The Herceptin is a targeted anticancer agent for target treatment of carcinomas overexpressing HER2, and has contributed greatly to improving the survival rate of patients in carcinomas of various stages, but when used alone, the complete remission rate is quite low. cited as a drawback.

On the other hand, scFv (Single Chain Variable Fragment) is an antibody fragment in which only the variable region of an antibody is recombined, and while maintaining antigen-specific targeting, it is advantageous for tissue penetration due to its small size, and is expected to be useful for research on next-generation targeted anticancer drugs.

Accordingly, the present inventors, based on the Herceptin-derived scFv, maintained the targeting of Herceptin to the HER2 protein, but developed scFv, anticancer peptide, and cell penetrating peptide to dramatically improve the problem of Herceptin's low complete remission rate. The present invention was completed by producing the recombinant fusion protein with an independently developed water-soluble protein expression system and confirming its effect.

KR 10-2020-0116394 A

An object of the present invention is to provide a novel anti-HER2 antibody-based dual-target anticancer agent capable of improving the low complete remission rate of existing anti-HER2 antibody-based anticancer agents while maintaining targeting to HER2.

In order to achieve the above object, the present invention provides an anti-HER2 scFv comprising the amino acid sequence represented by SEQ ID NO: 1; and a transcription factor CP2c target peptide comprising the amino acid sequence represented by SEQ ID NO: 3;

In addition, the present invention provides an expression vector containing a polynucleotide encoding the dual target protein.

In addition, the present invention provides a host cell transformed with the expression vector.

In addition, the present invention is directed to (1) transforming E. coli with an expression vector comprising a polynucleotide encoding a fusion protein comprising MBP, a TEV protease, a TEV protease cleavage site, and the dual target protein of claim 1 or claim 2 step; (2) culturing the E. coli to express the dual target protein; and (3) purifying the dual-target protein.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the dual target protein as an active ingredient.

The dual target protein according to the present invention includes an anti-HER2 scFv and a CP2c targeting peptide, so that HER2 targeting by the anti-HER2 scFv and CP2c targeting by the peptide can be simultaneously expressed. Accordingly, the dual target protein of the present invention can significantly improve the low complete remission rate seen in existing anti-HER2 antibody-based anticancer drugs while maintaining targeting to HER2.

In addition, the dual target protein of the present invention secures stability that can last for a long time in vivo, can selectively remove only various types of cancer cells, and can act on drug-resistant cancer cells.

1 is a DNA recombinant expressing fusion proteins of MBP-TEV-TEVsite-scFv (v24), MBP-TEV-TEVsite-scFv-ACP (v25) and MBP-TEV-TEVsite-scFv-ACP-iRGD (v26). (recombinant) is a schematic diagram.
2 is a schematic diagram showing the production and purification of soluble dual target proteins (including anti-Her2 scFv and CP2c target peptides) in E. coli by Ni-NTA chromatography.
Figure 3 is a diagram confirming the dual target protein expressed and purified from v24 (A), v25 (B) and v26 (C) recombinants by SDS-PAGE:
M: molecular weight marker (kDa);
T: total cellular protein;
P: insoluble fraction (Pelleted protein);
S: soluble fraction (Soluble protein);
BP: Binding pass;
W: washing pass; and
E: Protein eluted from Ni-NTA resin.
Figure 4a is a diagram showing the results of specificity and affinity analysis for HER2 antigen, and Figure 4b is a diagram showing the results of specificity and affinity analysis for BSA (control group):
Left: HER2 antigen;
right: BSA (control); and
KD: specific binding affinity.
Figure 5a is a diagram showing the results of specificity and affinity analysis for HER2 in SKOV3 cells (HER2 overexpressing cell line), and Figure 5b is a diagram showing the results of specificity and affinity analysis for HER2 in MDA-MB-231 (HER2-negative cell line) cells. is the diagram shown.
6 is a diagram showing a tumor growth curve over time in a SKOV3 transplanted mouse model.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.

In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

Throughout this specification, conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted 3-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (Nmethylglycine), etc. character codes are used. Amino acids also referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y, and valine: V.

The term "scFv (single chain fragment variable)" used in the present invention refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and is a single-chain form in which the VH and VL regions of an antibody are connected with a short peptide chain. refers to the antibody of The term “scFv” is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or otherwise understood from the context. This is obvious to a person skilled in the art.

Transcription factor CP2c is known to be overexpressed in various cancers, and according to a study by a research team in the US, it has been reported that suppressing the expression of CP2c in liver cancer cell lines inhibits growth, and overexpressing CP2c causes malignancy and metastasis of cancer. [Grant et al ., Antiproliferative small-molecule inhibitors of transcription factor LSF reveal oncogene addiction to LSF in hepatocellular carcinoma, Proc. Natl. Acad. Sci. 2012; 109(12): 4503-4508].

The CP2c target peptide of the present invention binds to the transcription factor CP2c and can interfere with DNA binding of CP2c.

dual target protein

In one aspect, the present invention provides an anti-HER2 scFv comprising the amino acid sequence represented by SEQ ID NO: 1; and a transcription factor CP2c target peptide comprising the amino acid sequence represented by SEQ ID NO: 3;

In one embodiment, the anti-HER2 scFv may be a Herceptin-derived scFv.

In one embodiment, the anti-HER2 scFv comprises a light chain variable region (VL) comprising the amino acid sequence represented by SEQ ID NO: 17 and a heavy chain variable region (VH) comprising the amino acid sequence represented by SEQ ID NO: 19 The variable region of the light chain and the variable region of the heavy chain may be connected by a linker comprising the amino acid sequence represented by SEQ ID NO: 21.

In one embodiment, the anti-HER2 scFv may further include 6xHis and c-Myc at the C-terminus, and may preferably include the amino acid sequence of SEQ ID NO: 15.

In one embodiment, a cell-penetrating peptide (CPP) may be coupled to the C-terminus of the CP2c target peptide to improve cell permeability. The cell membrane penetrating peptide may be internalizing RGD (iRGD), a peptide consisting of 9 amino acids (9aa) 'CRGDKGPDC (Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys, SEQ ID NO: 5)'.

In one embodiment, the dual target protein may be an anti-HER2 scFv, 6xHis, and c-Myc and CP2c target peptides linked sequentially. Preferably, the dual target protein may be sequentially linked anti-HER2 scFv, His6 and c-Myc, CP2c target peptide and cell membrane penetrating peptide (CPP).

In one embodiment, the dual target protein may include the amino acid sequence of SEQ ID NO: 7.

The dual target protein comprising the anti-HER2 scFv and the CP2c target peptide of the present invention, or the dual target protein comprising the anti-HER2 scFv, the P2c target peptide and the cell membrane penetrating peptide (CPP), is effective against HER2 by the anti-HER2 scFv. targeting, and CP2c targeting by peptides can be expressed simultaneously. Accordingly, the dual target protein of the present invention can significantly improve the low complete remission rate seen in existing anti-HER2 antibody-based anticancer drugs while maintaining targeting to HER2.

polynucleotide

In another aspect, the present invention relates to a polynucleotide encoding the dual target protein.

As used herein, the term “polynucleotide” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. A polynucleotide of the present invention may be a single strand of DNA or RNA. As used herein, the term "polynucleotide" includes DNA or RNA comprising modified bases and/or unique bases such as inosine. In the case of the DNA or RNA, it is obvious that various modifications can be made to serve known useful purposes. The term "polynucleotide" as used herein includes such chemically, enzymatically or metabolically modified polynucleotides.

Those skilled in the art will recognize that due to the degeneracy of the genetic code, the dual target protein may be encoded by several polynucleotides. That is, it is interpreted that the polynucleotide sequence encoding the dual target protein usable in the present invention includes a nucleotide sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 7. Preferably, the polynucleotide encoding the dual target protein of the present invention may include the nucleotide sequence represented by SEQ ID NO: 8.

In one embodiment, the polynucleotide consists of the nucleotide sequence represented by SEQ ID NO: 2 and encodes an anti-HER2 scFv; and a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4 and encoding a CP2c target peptide.

Preferably, the polynucleotide consists of the nucleotide sequence represented by SEQ ID NO: 2 and encodes an anti-HER2 scFv; a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 16 and encoding 6xHis and c-Myc; A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4 and encoding a CP2c target peptide; and a nucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 6 and encoding a cell membrane penetrating peptide.

In one embodiment, the anti-HER2 scFv may include a polynucleotide (SEQ ID NO: 18) encoding a light chain variable region (VL) and a polynucleotide (SEQ ID NO: 20) encoding a heavy chain variable region (VH). and a polynucleotide (SEQ ID NO: 22) encoding a linker connecting the variable region of the light chain and the variable region of the heavy chain.

The polynucleotide of the present invention is preferably an isolated polynucleotide. The term "isolated" polynucleotide refers to a polynucleotide that is substantially free of other nucleic acid sequences, such as, but not limited to, other chromosomal and extrachromosomal DNA and RNA. Isolated polynucleotides can be purified from host cells. To obtain isolated polynucleotides, conventional nucleic acid purification methods known to those skilled in the art can be used. The term also includes recombinant polynucleotides and chemically synthesized polynucleotides.

Expression system for dual target protein expression

Because scFv is smaller in size than general antibodies, it has a high penetration rate into tissues or cancer tissues while maintaining the antigen-specific targeting of the antibody, and can be mass-produced in various expression systems, including E. coli systems, and various molecular biological modifications are possible. It has the advantage of reducing production time and cost. However, when scFv (or dual target protein including scFv) is expressed in E. coli, most of it is expressed as insoluble, so there has been a problem in that the amount of protein expression that can be used functionally is very small. Accordingly, in the past, pelB and the like have been tried to improve the production yield of scFv (or dual target protein including scFv) in E. coli, but it has not been significantly effective, and when MBP is fused, scFv (or dual target protein including scFv) ), but was cumbersome and time-consuming because an additional purification process was required to separate pure scFv (or a dual target protein including scFv) from the fusion protein. Accordingly, the present inventors devised an expression system capable of purifying a soluble scFv (or a dual target protein including scFv) from E. coli at once without any additional process. According to the expression vector of the present invention and the protein production method using the expression vector, the target protein can be homogeneously produced in a soluble form in Escherichia coli, and since the target protein is cleaved by TEV protease expressed in cells, only the pure target protein can be produced. There is an effect that can be refined in one step.

expression vector

In another aspect, the present invention relates to an expression vector comprising a polynucleotide encoding a dual target protein including the anti-HER2 scFv and CP2c target peptide.

As used herein, the term "expression" means the production of a dual target protein in a cell.

As used herein, the term "expression vector" refers to a vector capable of expressing the dual target protein in a suitable host cell, and refers to a recombinant gene containing essential regulatory elements operably linked to express the gene insert. The expression vector refers to a vector including a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various regulatory sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions.

As used herein, the term “operably linked” refers to functional linkage between a nucleic acid expression control sequence and a polynucleotide encoding the dual target protein to perform a general function. For example, a promoter and a polynucleotide encoding the dual target protein may be operably linked to affect expression of the polynucleotide. Operational linkage with a recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and linking uses enzymes generally known in the art.

In one embodiment, the polynucleotide may include the nucleotide sequence of SEQ ID NO: 8.

In one embodiment, the expression vector can express a maltose-binding protein (MBP), a Tobacco etch virus protease (TEV protease), a TEV protease cleavage site, and a fusion protein including the dual target protein. .

In one embodiment, the fusion protein may be sequentially linked to MBP, TEV protease, TEV protease cleavage site, and the dual target protein.

In one embodiment, the MBP may comprise the amino acid sequence of SEQ ID NO: 9, the TEV protease may comprise the amino acid sequence of SEQ ID NO: 11, and the TEV protease cleavage site may comprise the amino acid sequence of SEQ ID NO: 13 .

In one embodiment, the fusion protein may further include 6xHis and c-Myc, and may preferably include the amino acid sequence of SEQ ID NO: 15.

In one embodiment, the expression vector consists of the nucleotide sequence represented by SEQ ID NO: 10, a polynucleotide encoding MBP; A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12 and encoding a TEV protease; A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 14 and encoding a TEV protease cleavage site; and a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8 and encoding the dual target protein.

In one embodiment, the expression vector consists of the nucleotide sequence represented by SEQ ID NO: 16, and may further include polynucleotides encoding 6xHis and c-Myc.

In one embodiment, the expression vector can solublely express a fusion protein comprising the dual target protein of the present invention and MBP in Escherichia coli, and the TEV protease in the fusion protein expressed in the cell is the TEV protease cleavage site (recognition site). ) is cleaved (self-cleavage), so pure dual-target proteins can be purified in one-step.

Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Suitable vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and may be prepared in various ways depending on the purpose. The vector's promoter may be constitutive or inducible. The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., and an α-amylase signal sequence and a subtilisin signal when the host is Bacillus sp. Sequences such as MFα signal sequence, SUC2 signal sequence, etc. can be used when the host is yeast, and insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used when the host is an animal cell. Not limited to this. The vector may also include a selectable marker for selecting host cells containing the vector and, in the case of a replicable expression vector, an origin of replication.

As used herein, the term "vector" refers to a delivery vehicle into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence. A nucleic acid sequence may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al ., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al. , In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).

In one embodiment, when constructing the vector, an expression control sequence such as a promoter, terminator, enhancer, etc., for membrane targeting or secretion, according to the type of host cell to produce the antibody. Sequences and the like can be selected appropriately and combined in various ways depending on the purpose.

In one embodiment, the expression vector is preferably prepared using any one vector selected from pET, pCold, pcDNA, pHIL-S1 and pRK793, preferably the pRK793 vector, in terms of effect.

host cell

In another aspect, the present invention relates to a host cell transformed with the expression vector.

In one embodiment, the host cell may be a bacterium, preferably E. coli.

Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used.

Methods for mass production of proteins

In another aspect, the present invention provides (1) an expression vector comprising MBP, a TEV protease, a TEV protease cleavage site, and a polynucleotide encoding a fusion protein comprising the dual target protein of claim 1 or claim 2 in Escherichia coli. transforming; (2) culturing the E. coli to express the dual target protein; and (3) purifying the dual-target protein.

In one embodiment, the fusion protein may be sequentially linked to MBP, TEV protease, TEV protease cleavage site, and the dual target protein.

In one embodiment, the dual target protein may be purified by Ni-NTA chromatography or a pull-down method using a tag.

In another aspect, the invention relates to a dual target protein produced by the method of the invention.

In another aspect, the present invention relates to the use of a dual target protein produced by the method of the present invention as an anti-cancer therapeutic agent.

pharmaceutical composition

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the dual target protein as an active ingredient.

Since the "dual target protein" of the present invention has already been described in detail, its description is omitted to avoid excessive duplication.

The term "prevention" of the present invention means anything that inhibits or delays the cancer.

The term "treatment" of the present invention, unless otherwise stated, means reversing the symptoms of, alleviating, inhibiting the progression of, or preventing the cancer.

The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the above active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, and lubricants. agents or flavoring agents may be used.

The dual target protein of the present invention can be formulated into a pharmaceutical preparation for cancer treatment. The purified protein may be dissolved in a conventional physiologically compatible aqueous buffer solution, to which optionally a pharmaceutical excipient may be added to provide a pharmaceutical preparation.

Such pharmaceutical carriers and excipients, as well as suitable pharmaceutical formulations, are known in the art (e.g., "Pharmaceutical Formulation Development of Peptides and Proteins", Frokjaer et al., Taylor & Francis (2000) or "Handbook of Pharmaceutical Excipients"). , 3rd edition, Kibbe et al., Pharmaceutical Press (2000)). In particular, the pharmaceutical composition containing the dual target protein of the present invention may be formulated in a lyophilized form or a stable liquid form. The dual target protein of the present invention can be lyophilized using various procedures known in the art. The lyophilized formulation is reconstituted prior to use by adding one or more pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline.

Formulations of the composition are delivered to a subject by any pharmaceutically suitable means of administration. A variety of delivery systems are known and can be used to administer the composition by any convenient route. Primarily, the compositions of the present invention are administered systemically. For systemic administration, the dual target protein of the present invention can be parenterally (e.g. intravenous, subcutaneous, intramuscular, intraperitoneal, intracerebral, intrapulmonary, intranasal or transdermal) delivery or enteral (e.g. oral, vaginal or rectal) are formulated for delivery. The most preferred routes of administration are intravenous and subcutaneous administration. These formulations may be administered continuously by infusion or bolus injection. Some formulations include sustained release systems.

The dual target protein of the present invention is administered to a patient in a therapeutically effective amount, which means a dosage sufficient to produce the desired effect while preventing or reducing the severity or spread of the condition or indication being treated without reaching a dosage that causes unacceptable side effects. do. The exact dosage depends on various factors such as indication, formulation, mode of administration, etc., and should be determined through preclinical and clinical trials for each indication.

The pharmaceutical composition of the present invention may be administered alone or in combination with other therapeutic agents. These agents may be included as part of the same medication. An example of such an agent is an anticancer drug such as Herceptin.

In one embodiment, the dual target proteins of the present invention are administered in an equivalent amount of 2 nanomolar each, and the specific dosage is 0.1 to 50 mg/kg, preferably 0.5 to 30 mg/kg, more preferably each. 1 to 10 mg/kg, more preferably 2 to 3 mg/kg each. When administration of the dual target protein is repeated more than once, the dosage may be the same or different each time.

Hereinafter, the present invention will be described in detail by examples, but the present invention is not limited by the following examples.

<Example>

Example 1: Synthesis of dual target protein - v26 (scFv-Myc-His6-ACP-CPP)

scFv (SEQ ID NO: 1), His6 and c-Myc (SEQ ID NO: 14), CP2c target peptide (ACP, SEQ ID NO: 3), and cell membrane penetrating peptide (CPP, SEQ ID NO: 5) are sequentially connected to the dual target protein (v26 , SEQ ID NO: 7) was synthesized.

The dual target protein is recombined by connecting the variable region of the light chain (SEQ ID NO: 16) and the variable region of the heavy chain (SEQ ID NO: 18) of the anti-HER2 antibody (Trastuzumab) with a linker sequence (SEQ ID NO: 20) to target HER2. (SEQ ID NO: 1), and His6 and c-Myc (SEQ ID NO: 14) were added for easy purification and labeling. CP2c targeting peptide was added twice to target CP2c (SEQ ID NO: 3), and a cell membrane penetrating peptide (SEQ ID NO: 5) was added to increase cell membrane permeability. In addition, a TEV protease cleavage sequence (SEQ ID NO: 13) was added for cleavage of the dual target protein from the platform.

For the above purpose, the amino acid sequence corresponding to the [TEVsite-scFv-Myc-His6-ACP-CPP] structure of the fusion protein was specified, and the nucleic acid sequence encoding the amino acid sequence was optimized to codons suitable for expression in E. coli. was decided.

In addition, for deletion of ACP and CPP in the future, an XbaI site and a SalI site adjacent to each sequence were added, and a stop codon was added to the end for termination. Then, for cloning, SacI site and PstI site were added to the ends, and the entire nucleic acid sequence was synthesized by commissioning Cosmogenetech Co., Ltd.

The synthesized nucleic acid sequence was subcloned into the restriction site SacI site and PstI site of pRK793 (Addgene; Plasmid #8827), an expression vector into which the MBP nucleic acid sequence was inserted, [MBP-TEVsite-scFv-Myc-His6-ACP- CPP] A vector for expression of the recombinant was prepared.

The nucleic acid sequence of the TEV protease was PCR from pRK793 by adding restriction site SacI site and XhoI site through forward primer (5'-AACGAGCTCGGGAGAAAGCTTG TTTAAGGGGCCG-3') and reverse primer (5'-GTCGAGCTCGAGGAACCATTCATGAGTTGAGTCGCTTC CTTAACTGG-3'), This was subcloned into the SacI site of the [MBP-TEVsite-scFv-Myc-His6-ACP-CPP] recombinant expression vector to express v26 encoding [MBP-TEV-TEVsite-scFv-Myc-His6-ACP-CPP]. vector was prepared.

The prepared v26 expression vector was transformed by applying heat shock at 42 ° C to E. coli BL21 (DE3) cells, spread on LB solid medium (Ampicillin 100ug/mL), cultured for 15 hours, and then excellent strains were selected. Expression of the selected superior strain was induced again in 500 mL of LB liquid medium (Ampicillin 100ug/mL) under conditions of O.D.600=1.0, IPTG 0.2 mM, and 15 ° C for 16 hr, and about 3 g of cells were obtained from the culture medium. . Thereafter, the obtained cells were suspended in PBS to disrupt the cells, the supernatant was applied to a Ni-NTA column, the column was washed with 25 mM imidazole, and v26 protein was eluted with 500 mM imidazole. E. coli transformed with the v26 expression vector expressed the protein of [MBP-TEV-TEVsite-scFv-Myc-His6-ACP-CPP], which was cleaved in E. coli and double-targeted protein v26 (scFv-Myc-His6- ACP-CPP) was purified. Important samples in the protein purification process were identified by SDS-PAGE (Fig. 3, C).

Example 2: Synthesis of dual target protein - v25 (scFv-Myc-His6-ACP)

A dual target protein (v25, SEQ ID NO: 23) was produced in which scFv (SEQ ID NO: 1), His6 and c-Myc (SEQ ID NO: 14), and CP2c target peptide (ACP, SEQ ID NO: 3) were sequentially connected.

From the v26 expression vector expressing [MBP-TEV-TEVsite-scFv-Myc-His6-ACP-CPP], two SalI sites adjacent to CPP were subcloned to [MBP-TEV-TEVsite-scFv-Myc-His6 -ACP] was prepared for expression of v25.

The prepared v25 expression vector was transformed by applying heat shock to E. coli BL21 (DE3) cells at 42°C, spread on LB solid medium (Ampicillin 100ug/mL) and cultured for 15 hours, and then superior strains were selected. Expression of the selected superior strain was induced again in 500 mL of LB liquid medium (Ampicillin 100ug/mL) under conditions of O.D.600 = 1.0, IPTG 0.2mM, and 15°C for 16 hr, and about 3 g of cells were obtained from the culture medium. Thereafter, the obtained cells were suspended in PBS to disrupt the cells, the supernatant was applied to a Ni-NTA column, the column was washed with 25 mM imidazole, and v25 protein was eluted with 500 mM imidazole. E. coli transformed with the v25 expression vector expressed the protein of [MBP-TEV-TEVsite-scFv-Myc-His6-ACP], which was cleaved in E. coli to form the dual target protein v25 (scFv-Myc-His6-ACP) only has been refined. Important samples in the protein purification process were identified by SDS-PAGE (FIG. 3, B).

Comparative Example: v24 (scFv-Myc-His6)

A scFv (SEQ ID NO: 1) and a protein (v24) linked to 6xHis and c-Myc (SEQ ID NO: 14) were synthesized.

From the v25 expression vector expressing [MBP-TEV-TEVsite-scFv-Myc-His6-ACP], two XbaI sites adjacent to the ACP were subcloned to obtain [MBP-TEV-TEVsite-scFv-Myc-His6]. A vector for expressing the encoding v24 was prepared.

The prepared v24 expression vector was transformed by applying heat shock to E. coli BL21(DE3) cells at 42°C, spread on LB solid medium (Ampicillin 100ug/mL) and cultured for 15 hours, and then superior strains were selected. Expression of the selected superior strain was induced again in 500 mL of LB liquid medium (Ampicillin 100ug/mL) under conditions of O.D.600 = 1.0, IPTG 0.2mM, and 15°C for 16 hr, and about 3 g of cells were obtained from the culture medium. Thereafter, the obtained cells were suspended in PBS to disrupt the cells, the supernatant was applied to a Ni-NTA column, the column was washed with 25 mM imidazole, and v25 protein was eluted with 500 mM imidazole. E. coli transformed with the v25 expression vector expressed the v24 protein of [MBP-TEV-TEVsite-scFv-Myc-His6], which was cleaved in E. coli and only the anti-HER2 scFv protein v24 (scFv-Myc-His6) has been refined Important samples in the protein purification process were identified by SDS-PAGE (Fig. 3, A).

<Test Example>

Test Example 1: Preparation of fusion protein for production of dual target protein and plasmid encoding the same

The present inventors fused MBP (maltose-binding protein) to scFv in order to express and produce scFv (single chain variable fragment), which is expected to replace existing antibody therapeutics, in a soluble form in E. coli. To remove scFv, a plasmid was constructed to contain TEV protease (Tobacco etch virus protease) and its recognition site, and it was expressed and purified by Ni-NTA chromatography.

Specifically, as long as the proteins of Example 1, Example 2 or Comparative Example including MBP (SEQ ID NO: 8), TEV protease (SEQ ID NO: 10), and TEV recognition site (SEQ ID NO: 12) are sequentially linked A plasmid was constructed to express the stranded fusion protein (FIG. 1). When the plasmid encoding the fusion protein of the present invention is expressed in Escherichia coli, it is expressed in the form of soluble v24, v25 or v26 in FIG. It can be purified at once by the Ni-NTA chromatography method (FIG. 2).

Test Example 2: Confirmation of protein expression and purification according to the expression platform

The MBP-TEV protease-TEV recognition site of the present invention prepared in Test Example 1-a plasmid encoding a fusion protein in which the protein v26 of Example 1 is sequentially linked, and the MBP-TEV protease-TEV recognition site-Example 2 A plasmid encoding a fusion protein in which protein v25 is sequentially linked, and a plasmid encoding a fusion protein in which MBP-TEV protease-TEV recognition site-comparative protein v24 is sequentially linked are expressed in E. coli (with/without IPTG) and Ni It was purified by Ni-NTA chromatography using -NTA resin, and the purified proteins were confirmed using SDS-PAGE (see FIG. 3).

As a result, in the case of the v26 protein of Example 1, the v26 protein cleaved in cells by TEV protease was present in the soluble fraction (S), and the v26 protein was obtained by Ni-NTA chromatography or pull-down method. It was found to be pure and purified (Fig. 3C). In addition, the v25 protein of Example 2 (FIG. 3B) and the v24 protein of Comparative Example (FIG. 3C) were also present in the soluble fraction (S) as in the case of the v26 protein, and Ni-NTA chromatography or pull-down method It has been shown to be pure purified at once.

On the other hand, when a plasmid encoding only v24 (His6 and c-Myc attached to scFv) without MBP-TEV protease-TEV recognition site was expressed in E. coli, the v24 was mostly expressed as insoluble and present in the pellet (P) , it was shown that only a very small amount was solublely expressed.

Test Example 3: Binding affinity analysis of purified protein

3-1: Analysis of binding affinity to HER2 protein

The binding affinity of the v25 (scFv-ACP), v26 (scFv-ACP-CPP) and v24 (scFv) proteins expressed and purified by the platform of the present invention in Test Example 2 was examined.

Specifically, HER2 (Human Epidermal growth factor receptor2) protein (Acro biosystem, HE2-H5225) or BSA (control) was mixed with coating buffer (200 mM Na ₂ Co ₃ , 200 mM NaHCo ₃ , pH 9.6) at 50 ng/mL. After dilution, 100 ul each was dispensed into a 96-well ELISA plate and adsorbed overnight at 4 °C. Thereafter, unadsorbed antigens were removed by washing three times with PBS-T (PBS, 0.05% Tween20), and 200 ul of 5% skim milk (PBS, 5% w/v skim milk powder) was added to each well at room temperature. and blocked for 1 hour before removal. Each well was treated with 100 ul of the v25, v26 and v24 proteins expressed and purified in Test Example 2 at a concentration of 0.1 pM to 400 nM, respectively, and incubated at room temperature for 1 hour. Unbound antibodies were removed by washing three times with PBS-T (PBS, 0.05% Tween20), and ProteinL-HRP (Genscript, M00098) (in PBS), which detects VL, was added to each well at a concentration of 50 ng/mL. 100 ul each at room temperature and incubated for 1 hour. Wash three times with PBS-T (PBS, 0.05% Tween20) to remove unbound ProteinL-HRP, and treat each well with 100 ul of TMB containing 1.5% H ₂ O ₂ at room temperature for 30 minutes. A color reaction was induced. Then, 100 ul of 1M HCL was added to each well to stop the reaction, and absorbance was measured at 450 nm. Each experiment was repeated three times, and the measured values and standard deviations at each concentration were plotted using the Prism8 program. In addition, the specific binding affinity (KD value) was determined using ELISA and LSF (least-squares fit) (see FIG. 4).

Referring to FIG. 4, it can be confirmed that the v25, v26 and v24 proteins expressed and purified in Test Example 2 each show specific binding affinity to the antigen HER2, but do not have binding affinity to the control protein BSA. there is. In addition, the v25 and v26 proteins of Example showed higher binding affinity to HER2 than the v24 protein of Comparative Example, and in particular, the v26 protein of Example 1 had higher binding to HER2 than the v25 protein of Example 2. It has been shown to have affinity.

3-2: Analysis of binding affinity to HER2 overexpressing cell lines

The binding affinities of the v25 (scFv-ACP), v26 (scFv-ACP-CPP) and v24 (scFv) proteins expressed and purified by the platform of the present invention in Test Example 2 were examined using the HER2 overexpressing cell line SKOV3. .

Specifically, the SKOV-3 cell line (HER2 overexpressing cell line) or MDA-MB-231 cell line (HER2 negative cell line) cultured in a petri dish in a 37 ° C., 5% CO ₂ environment was washed and 1% BSA (PBS, 1% Bovine Serum Albumin) and dispensed into 96-well U-type bottom plates in 10 ⁶ units. Then, each well was treated with the v25, v26 and v24 proteins expressed and purified in Test Example 2 at a concentration of 0.1 pM to 500 nM, respectively, and incubated at 4° C. for 30 minutes. The cells were centrifuged at 4 °C and 500 g for 5 minutes, resuspended in 1% BSA (PBS, 1% Bovine Serum Albumin), and washed twice with 1% BSA (PBS, 1% Bovine Serum Albumin). Then, ProteinLFITC (Acrobiosystems, RPL-PF141) was diluted to 5 ug/mL in 1% BSA (PBS, 1% Bovine Serum Albumin), treated with 100 ul per well, and incubated for 30 minutes at 4 ° C in a dark room. did After washing twice with 1% BSA (PBS, 1% Bovine Serum Albumin), the cells were centrifuged at 4 °C and 500 g for 5 minutes and resuspended in 100 ul of PBS. Thereafter, flow cytometry data were derived using a FACSCalibur flow cytometer (BD, USA), and measured values and standard deviations were plotted using the Prism8 program. In addition, the specific binding affinity (KD value) was determined using flow cytometry and least-squares fit (LSF) (see FIG. 5).

Referring to FIG. 5, it was confirmed that all of the v26, v25, and v24 proteins had specific binding affinity to the HER2 overexpressing cell line SKOV3, and no binding affinity to the control cell line MDA-MB-231. there is. In addition, it can be seen that the binding affinity to the HER2 overexpressing cell line SKOV3 is in the order of v26, v25 and v24, respectively.

Test Example 4: Tumor growth inhibitory effect in xenograft model

Female Balb/c nude mice were 6 weeks old and purchased from Orient Co. (Seongnam, Gyeonggi-do). Thereafter, 3 animals per cage were randomly housed regardless of the experimental group. Rats were bred under controlled laboratory conditions (temperature 22±2 °C, humidity 55±5%, 12-hour light/dark cycle) by supplying sufficient standard feed and water. SKOV3 cells were prepared by culturing in a culture dish at 37 ℃, 5% CO ₂ environment, and 3 × 10 ⁶ cells per animal were mixed with Matrigel and injected subcutaneously. Thereafter, when the tumor volume reached 200 mm (width, length, height), the purified protein samples v26, v25, and v24 were respectively iv injected. Protein samples purified from each group were injected with a total molarlity of 6 nanomole, and the concentration of all samples was 100 uM, and a volume of around 100 ul was injected. Then, 100 ul of PBS was injected into the control group. Injection lasted for 18 days, and after that, they were observed and raised without injection for 10 days. During the injection period and the observation period, the size of the tumor was measured once a day, and the measured values and standard deviations of the experimental animals in each group were plotted using the Prism8 program. During the experiment, no deaths or abnormal signs were observed in all subjects including the control group.

Referring to Figure 6, it can be seen that the protein (v25 and v26) of the example has a significantly higher tumor growth inhibitory effect than the protein (v24) of the comparative example. The protein (v24) of the comparative example rapidly increased in tumor size after sample injection was stopped, and the size of the tumor was the same as that of the control group on the 6th to 7th days after the injection of the sample was stopped. On the other hand, the proteins of Example did not rapidly increase in tumor size even after sample injection was stopped, and in particular, the protein of Example 1 hardly increased in tumor size.

These results indicate that the dual target protein including the anti-HER2 scFv and the CP2c target peptide of the present invention can be usefully used as a novel anticancer treatment.

Although the present invention has been described in terms of the preferred embodiments mentioned above, various modifications and variations are possible without departing from the spirit and scope of the invention. The appended claims also cover such modifications and variations as fall within the subject matter of this invention.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Double-targeting protein comprising anti-HER2 scFv and peptide for targeting CP2c and use thereof <130> HP10421 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211 > 246 <212> PRT <213> Artificial Sequence <220> <223> Anti-HER2 scFv <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ser Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr 165 170 175 Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly 210 215 220 Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Ala Ser 245 <210> 2 <211> 738 <212> DNA <213> Artificial Sequence <220> <223> Anti-HER2 scFv <400 > 2 gacattcaaa tgacgcaatc gcctagttcg ctgagtgcga gtgtcggcga ccgcgtgacc 60 ataacgtgtc gggccagtca ggatgtaaat accgccgtgg cttggtacca gcagaagccg 120 ggcaaggcgc ctaagctgct tatatattcc gcttcctttc tgtacagc gg tgtgccatct 180 cgtttctcag gctcgcggtc tgggacggac ttcacactta caatctcctc tcttcagccg 240 gaggatttcg caacatatta ctgtcagcaa cactatacta caccccccac ttttggccaa 300 gggacgaaag tcgagatcaa acgttcaggg ggaggcggtt ctggcggggg aggttcggga 360 ggaggtggaa gcgaagtaca actggtggag agcggtggtg gtttagttca gccgggcggt 540 tatacacgtt atgccgactc cgtgaaagga agatttacta tcagtgcaga tacgtccaaa 600 aacacggcct accttcaaat gaactcactg cgcgcagaag acacggcagt ttattactgt 660 tcgcgttggg gtggcgatgg gttttatgcc atggattatt gggggcaggg tacccttgtg 720 accgtttcct ccgcctcc 738 <210> 3 <211> 1 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide for targeting CP2c <400> 3 Asn Tyr Pro Gln Arg Pro Gly Gly Asn Tyr Pro Gln Arg Pro 1 5 10 <210> 4 <211> 42 <212> DNA < 213> Artificial Sequence <220> <223> Peptide for targeting CP2c <400> 4 aattacccac agagaccagg aggtaattac ccccagcgtc cg 42 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CPP (cell -penetrating peptide) <400> 5 Cys Arg Gly Asp Lys Gly Pro Asp Cys 1 5 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CPP (cell-penetrating peptide) < 400> 6 tgtcgtggcg ataaaggccc ggactgt 27 <210> 7 <211> 295 <212> PRT <213> Artificial Sequence <220> <223> v26 (Double-targeting protein) <400> 7 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ser Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr 165 170 175 Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly 210 215 220 Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Ala Ser Gly Ser Glu Gln Lys Leu Ile Ser Glu Glu 245 250 255 Asp Leu His His His His His Gly Ser Ser Arg Asn Tyr Pro Gln 260 265 270 Arg Pro Gly Gly Asn Tyr Pro Gln Arg Pro Ser Arg Val Asp Cys Arg 275 280 285 Gly Asp Lys Gly Pro Asp Cys 290 295 <210> 8 <211> 888 <212> DNA <213> Artificial Sequence <220> <223> v26 (Double-targeting protein) <400> 8 ggtgacattc aaatgacgca atcgcctagt tcgctgagtg cgagtgtcgg cgaccgcgtg 60 accataacgt gtcgggccag tcaggatgta aataccgccg tggcttggta ccagcagaag 120 ccgggcaagg cgcctaagct gcttatatat tccgcttcct ttctgtacag cggtgtgcca 180 tctcgtttct caggctcgcg gtctgggacg gacttcacac ttacaatctc ctctcttcag 240 ccggaggatt tcgcaacata ttactgtcag caacactata ctacaccccc cacttttggc 300 caagggacga aagtcgagat caaacgttca gggggaggcg gttct ggcgg gggaggttcg 360 ggaggaggtg gaagcgaagt acaactggtg gagagcggtg gtggtttagt tcagccgggc 420 ggttctttga gattaagctg tgctgccagc ggttttaata tcaaggatac ttatattcac 480 tgggtccgtc aagctccagg aaaaggt ttg gaatgggtgg ctagaatcta ccctaccaac 540 gggtatacac gttatgccga ctccgtgaaa ggaagattta ctatcagtgc agatacgtcc 600 aaaaacacgg cctaccttca aatgaactca ctgcgcgcag aagacacggc agtttattac 660 tgttcgcgtt ggggtggcga tgggttttat gccatggatt attgggggca gggtaccctt 720 gtgaccgttt cctccgcctc cggatccgag cagaaattga tttcagaaga ggatct artificial Sequence <220> <223> MBP <400> 9 Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser 370 <210> 10 <211> 1113 <212> DNA <213> Artificial Sequence <220> <223> MBP <400> 10 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgtt ac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtact t cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caa accgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgccccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcg 1113 <210> 11 <21 1> 242 <212> PRT <213> Artificial Sequence <220> <223> TEV protease <400> 11 Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 1 5 10 15 Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 20 25 30 Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35 40 45 Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe 50 55 60 Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg 65 70 75 80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln 85 90 95 Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val 100 105 110 Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125 Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 150 155 160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 165 170 175 Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180 185 190 Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser 195 200 205 Val Leu Trp Gly Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro 210 215 220 Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Gly Ser Ser Ser Ser 225 230 235 240 Ser Gly <210> 12 <211> 726 <212> DNA < 213> Artificial Sequence <220> <223> TEV protease <400> 12 ggagaaagct tgtttaaggg gccgcgtgat tacaacccga tatcgagcac catttgtcat 60 ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg gtattggatt tggtcccttc 120 atcattacaa aca agcactt gtttagaaga aataatggaa cactgttggt ccaatcacta 180 catggtgtat tcaaggtcaa gaacaccacg actttgcaac aacacctcat tgatgggagg 240 gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa gctgaaattt 300 agagagccac aaagggaaga gcgcatatgt cttgtgacaa ccaacttcca aactaagagc 360 atgtctagca tggtgtcaga cactagttgc acattccctt catctgatgg catattctgg 420 aagcattgga ttcaaaccaa ggatgggcag tgtggcagtc cattagtatc aactagagat 4 80 gggttcattg ttggtataca ctcagcatcg aatttcacca acacaaacaa ttatttcaca 540 agcgtgccga aaaacttcat ggaattgttg acaaatcagg aggcgcagca gtgggttagt 600 ggttggcgat taaatgctga ctcagtattg tgggggggcc ataaagtttt cat ggtgaaa 660 cctgaagagc cttttcagcc agttaaggaa gcgactcaac tcatgaatgg ttcctcgagc 720 tcggga 726 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> TEV protease cleavage site <400> 13 Glu Asn Leu Tyr Phe Gln Gly 1 5 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TEV protease cleavage site <400> 14 gaaaatttat atttccaggg t 21 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> His6 and c-Myc <400> 15 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu His His His His His His 1 5 10 15 <210> 16 <211> 48 <212> DNA <213> Artificial Sequence <220> < 223> His6 and c-Myc <400> 16 gagcagaaat tgatttcaga agaggatctg caccatcatc atcatcac 48 <210> 17 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL of anti-HER2 scFv <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ser 100 105 <210> 18 <211> 327 <212> DNA <213> Artificial Sequence <220 > <223> VL of anti-HER2 scFv <400> 18 gacattcaaa tgacgcaatc gcctagttcg ctgagtgcga gtgtcggcga ccgcgtgacc 60 ataacgtgtc gggccagtca ggatgtaaat accgccgtgg cttggtacca gcagaagccg 120 ggcaaggcgc ctaagct gct tatatattcc gcttcctttc tgtacagcgg tgtgccatct 180 cgtttctcag gctcgcggtc tgggacggac ttcacactta caatctcctc tcttcagccg 240 gaggatttcg cacacatatta ctgtcagcaa cactatacta caccccccac ttttggccaa 30 0 gggacgaaag tcgagatcaa acgttca 327 <210> 19 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> VH of anti-HER2 scFv <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 <210> 20 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> VH of anti-HER2 scFv <400> 20 gaagtacaac tggtggagag cggtggtggt ttagttcagc cgggcggttc tttgagatta 60 agctgtgctg ccagcggttt taatatcaag gatacttata ttcactgggt ccgtcaagct 120 ccaggaaaag gtttggaatg ggtggctaga atctacccta ccaacgggta tacacgttat 180 gccgactccg tgaaaggaag attactatc agtgcagata cgtccaaaaa cacggcctac 240 cttcaaatga actcactgcg cgcagaagac acggcagttt attactgttc gcgttggggt 300 ggcgatgggt tttatgccat ggattattgg gggcagggta cccttgtgac cgtttcctcc 360 gcctcc 366 <210> 21 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker Connecting VL and VH <400> 21 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 22 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Linker Connecting VL and VH <400> 22 gggggaggcg gttctggcgg gggaggttcg ggaggaggtg gaagc 45 < 210> 23 <211> 286 <212> PRT <213> Artificial Sequence <220> <223> v25 (Double-targeting protein) <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ser Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr 165 170 175 Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly 210 215 220 Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Ala Ser Gly Ser Glu Gln Lys Leu Ile Ser Glu Glu 245 250 255 Asp Leu His His His His His Gly Ser Ser Arg Asn Tyr Pro Gln 260 265 270 Arg Pro Gly Gly Asn Tyr Pro Gln Arg Pro Ser Arg Val Asp 275 280 285 <210> 24 <211> 861 <212> DNA <213> Artificial Sequence <220> <223> v25 (Double -targeting protein) <400> 24 ggtgacattc aaatgacgca atcgcctagt tcgctgagtg cgagtgtcgg cgaccgcgtg 60 accataacgt gtcgggccag tcaggatgta aataccgccg tggcttggta ccagcagaag 120 ccgggcaagg cgcctaagct gcttatatat tccgct tcct ttctgtacag cggtgtgcca 180 tctcgtttct caggctcgcg gtctgggacg gacttcacac ttacaatctc ctctcttcag 240 ccggaggatt tcgcaacata ttactgtcag caacactata ctacaccccc cacttttggc 300 caagggacga aagtcgagat ca aacgttca gggggaggcg gttctggcgg gggaggttcg 360 ggaggaggtg gaagcgaagt acaactggtg gagagcggtg gtggtttagt tcagccgggc 420 ggttctttga gattaagctg tgctgccagc ggttttaata tcaaggatac ttatattcac 480 tgggtccgtc aagctccagg aaaaggtttg gaatgggtgg ctagaatc at tgggggca gggtaccctt 720 gtgaccgttt cctccgcctc cggatccgag cagaaattga tttcagaaga ggatctgcac 780 catcatcatc atcacggatc ctctagaaat tacccacaga gaccaggagg taattacccc 840cagcgtccgt ctagagtcga c 861

Claims (16)

an anti-HER2 scFv comprising the amino acid sequence represented by SEQ ID NO: 1; and a peptide targeting the transcription factor CP2c comprising the amino acid sequence represented by SEQ ID NO: 3. According to claim 1,
The CP2c target peptide is a dual target protein, characterized in that a cell membrane penetrating peptide (CPP) comprising the amino acid sequence represented by SEQ ID NO: 5 is bound to the C-terminus. An expression vector comprising a polynucleotide encoding the dual target protein of claim 1 or 2. According to claim 3,
The expression vector, characterized in that the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8. According to claim 3,
The expression vector is characterized by expressing a fusion protein comprising a maltose-binding protein (MBP), a Tobacco etch virus protease (TEV protease), a TEV protease cleavage site, and the dual target protein Expression vector. According to claim 5,
The fusion protein is an expression vector, characterized in that MBP, TEV protease, TEV protease cleavage site and dual target protein are sequentially connected. According to claim 5,
The expression vector characterized in that the MBP comprises the amino acid sequence represented by SEQ ID NO: 9. According to claim 5,
The expression vector characterized in that the TEV protease comprises the amino acid sequence represented by SEQ ID NO: 11. According to claim 5,
The expression vector characterized in that the TEV protease cleavage site comprises the amino acid sequence represented by SEQ ID NO: 13. The method of claim 5, wherein the expression vector
A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 10 and encoding MBP;
A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12 and encoding a TEV protease;
A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 14 and encoding a TEV protease cleavage site; and
An expression vector comprising a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8 and encoding the dual target protein. A host cell transformed with the expression vector of claim 5. According to claim 11,
The host cell is a host cell, characterized in that Escherichia coli. (1) transforming E. coli with an expression vector comprising a polynucleotide encoding a fusion protein comprising MBP, a TEV protease, a TEV protease cleavage site, and the dual target protein of claim 1 or 2;
(2) culturing the E. coli to express the dual target protein; and
(3) purifying the dual-target protein; a method for mass-producing dual-target protein, including. According to claim 13,
The fusion protein is a mass production method of a dual-target protein, characterized in that MBP, TEV protease, TEV protease cleavage site and dual-target protein are sequentially connected. According to claim 13,
The purification is a method for mass production of dual target proteins, characterized in that carried out by Ni-NTA chromatography method or pull-down method. A pharmaceutical composition for preventing or treating cancer comprising the dual target protein according to claim 1 or 2 as an active ingredient.

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