KR20240113641A - The tumor-targeting antibody fusion protein platform comprising tumor-targeting carrier, MMP cleavage site, anti-cancer peptide and cell penetrating peptide - Google Patents

Abstract

The present invention relates to a novel tumor-targeting antibody fusion protein platform which fuses an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide with a tumor-targeting carrier comprising an antibody or a fragment thereof that recognizes a tumor-associated antigen. The tumor-targeting antibody fusion protein according to the present invention can simultaneously express targeting by the tumor-targeting carrier and tumor-targeting by the peptide by including the tumor-targeting carrier, the MMP cleavage sequence, the anticancer peptide, and the cell-penetrating peptide. Accordingly, the tumor-targeting antibody fusion protein of the present invention can significantly improve the low complete response rate observed in existing antibody-based anticancer agents while maintaining targeting to a tumor-associated antigen. In addition, the tumor-targeting antibody fusion protein of the present invention secures stability that can be sustained for a long time in vivo, can selectively remove only various types of cancer cells, and can act on drug-resistant cancer cells.

Description

The tumor-targeting antibody fusion protein platform comprising a tumor-targeting carrier, MMP cleavage site, anti-cancer peptide and cell penetrating peptide }

The present invention relates to a new tumor-targeting antibody fusion protein platform that fuses an MMP cleavage sequence, an anti-cancer peptide, and a cell-penetrating peptide with a tumor-targeting delivery vehicle such as an antibody that recognizes a tumor-related antigen or an antigen-binding fragment thereof.

Monoclonal antibodies are antibodies with a single affinity and are widely used as antibody treatments such as targeted anticancer drugs by specifically targeting specific antigens or cells in living organisms. However, when used alone, the antibody treatment has a problem of low complete remission rate.

Accordingly, the present inventors maintained the targetability for tumor-related antigens based on tumor targeting carriers such as monoclonal antibodies or antigen-binding fragments thereof, but overexpressed cancer cells to improve the problem of low complete remission rate. We developed a tumor-targeting antibody fusion protein that fused a sequence (MMP site) that is cleaved by the protein MMP (Matrix metalloproteinase), an anti-cancer peptide, and a cell-penetrating peptide. The tumor-targeting antibody fusion protein targets cancer cells, but The present invention was completed by confirming that it has an excellent ability to kill cancer cells by being specifically cut on the external surface.

As an example of a tumor-associated antigen, the Her2/neu (ErbB2) gene encodes a 185 kDa transmembrane glycoprotein, which is a member of the family of epidermal growth factor receptors (EGFR). The Her2 protein consists of an extracellular domain of 620 amino acid residues, a transmembrane domain of 23 amino acid residues, and an intracellular domain of 490 amino acid residues with tyrosine kinase activity (Akiyama T, et al ., Science, 232 ( 4758):1644-1646(1986)). The HER2 gene is used in breast cancer, colon cancer, gallbladder cancer, cholangiocarcinoma, bladder cancer, ovarian cancer, uterine cancer, pancreas cancer, prostate cancer, brain cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, liver cancer, lung cancer, and head and neck cancer. , overexpression is observed in esophageal cancer and stomach cancer.

As an example of a monoclonal antibody, anti-HER2 antibodies with various properties targeting the HER2 gene have been disclosed in many literatures: Tagliabue et al. , Int. J. Cancer 47:933-937 (1991); McKenzie et al ., Oncogene 4:543-548 (1989); Maier et al ., Cancer Res. 51:5361-5369 (1991); Bacus et al ., Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al ., PNAS (USA) 88:8691-8695 (1991); Bacus et al ., Cancer Research 52:2580-2589 (1992); Xu et al ., Int. J. Cancer 53:401-408 (1993); WO94/00136; Kasprzyk et al ., Cancer Research 52:2771-2776 (1992); Hancock et al ., Cancer Res. 51:4575-4580 (1991); Shawver et al. , Cancer Res. 54:1367-1373 (1994); Arteaga et al ., Cancer Res. 54:3758-3765 (1994); Harwerth et al ., J. Biol. Chem. 267:15160-15167 (1992); US Pat. No. 5,783,186; Kao et al ., US Publ. No. 2009/0285837 (2009); Ross et al ., The Oncologist 8:307-325 (2003) and Klapper et al ., Oncogene 14:2099-2109 (1997).

The present invention relates to a tumor-targeting delivery vehicle such as an antibody or antigen-binding fragment thereof, and an anti-cancer composition containing a tumor-targeting delivery vehicle, an MMP cleavage sequence, an anti-cancer peptide, and a cell-penetrating peptide.

The present invention will be described in more detail through examples using the HER2 antigen binding fragment below. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

KR 10-2021-0157688 A KR 10-2020-0116394 A

The purpose of the present invention is to provide a tumor-targeting antibody fusion protein platform that can improve the low complete response rate of existing antibody-based anticancer drugs while maintaining targetability for tumor-related antigens.

In order to achieve the above object, the present invention provides a tumor targeting carrier; MMP cleavage site that is cleaved by MMP (Matrix metalloproteinase); An anti-cancer peptide and a cell-penetrating peptide are sequentially linked, the tumor-targeting delivery vehicle is an antibody containing an amino acid sequence that specifically binds to a tumor-related antigen, or an antigen-binding fragment thereof, and the MMP cleavage sequence is MMP (Matrix metalloproteinase) It provides a tumor-targeting antibody fusion protein, which includes an amino acid sequence cleaved by ).

Additionally, the present invention provides an expression vector containing a polynucleotide encoding the tumor-targeting antibody fusion protein.

Additionally, the present invention provides host cells transformed with the expression vector.

In addition, the present invention includes the steps of (1) transforming E. coli with an expression vector containing a polynucleotide encoding MBP, TEV protease, a TEV protease cleavage sequence, and a second fusion protein containing the tumor-targeting antibody fusion protein; (2) culturing the E. coli to express a tumor-targeting antibody fusion protein; and (3) purifying the tumor-targeting antibody fusion protein.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the tumor-targeting antibody fusion protein as an active ingredient.

The tumor-targeting antibody fusion protein according to the present invention contains a tumor-targeting carrier, an MMP cleavage sequence, an anti-cancer peptide, and a cell-penetrating peptide, and thus can simultaneously express targeting by the tumor-targeting carrier and tumor targeting by the peptide. Accordingly, the tumor-targeting antibody fusion protein of the present invention can significantly improve the low complete response rate seen in existing antibody-based anticancer drugs while maintaining targeting against tumor-related antigens.

In addition, the tumor-targeting antibody fusion protein of the present invention ensures long-term stability in vivo, can selectively eliminate various types of cancer cells, and can also act on drug-resistant cancer cells.

Figure 1 is a summary diagram briefly showing the tumor-targeting antibody fusion protein platform of the present invention.
Figure 2 shows the results of testing the cell death ability of a fusion protein in which various peptides (iRGD, dNP2, mdNP2, mmdNP2, or 9R) expected to have cell-penetrating ability are fused to the anti-cancer peptide (ACP or KLAK) of the present invention. . Figure 2a is a result of testing the apoptotic ability of the fusion protein against cancer cells, and Figure 2b is a graph showing the quantification of the apoptotic ability of the fusion protein against cancer cells.
Figure 3 is a comparative example of the present invention, omitting the MMP cleavage sequence of the present invention, and showing the results of testing the effect of a tumor-targeting antibody fusion protein in which a tumor-targeting delivery vehicle, an anti-cancer peptide, and a cell-penetrating peptide are fused. Figure 3a is a schematic diagram showing the fusion protein, Figure 3b is the result of confirming the expression of the fusion protein by SDS-PAGE, and Figure 3c is a graph showing the binding affinity of the fusion protein to the target antigen. , Figure 3d is a result showing the apoptotic effect of the fusion protein on cancer cells.
Figure 4 is an example of the present invention, showing the results of testing the effect of a tumor-targeting antibody fusion protein fused with the tumor-targeting delivery vehicle, MMP cleavage sequence, anticancer peptide, and cell-penetrating peptide of the present invention. Figure 4a is a diagram confirming the fusion proteins (D303 and D305) by SDS-PAGE, Figure 4b is a result showing the apoptotic effect of the fusion proteins (D303 and D305) on cancer cells, and Figure 4c is a diagram showing the fusion proteins (D303 and D305) D301, D302, D303 and D304) were confirmed by SDS-PAGE, and Figure 4d shows the results showing the apoptotic effect of the fusion proteins (D301, D302, D303 and D304) on cancer cells.
Figure 5 is an example of the present invention, showing the results of testing the effect of a tumor-targeting antibody fusion protein fused with the tumor-targeting delivery vehicle, MMP cleavage sequence, anticancer peptide, and cell-penetrating peptide of the present invention. Figures 5a and 5b show the results of confirming the expression of the fusion proteins (D310, D311, D312, D313, D314, D315, D316) by SDS-PAGE, and Figure 5c shows the results of confirming the expression of the fusion proteins (D310, D311, D312, D313, A diagram showing the results of specificity and affinity analysis for HER2 antigen or HSA (serum albumin) protein (D314, D315, D316), and Figure 5d shows the fusion proteins (D310, D311, D312, D313, D314, D315, D316) This result shows the apoptosis effect on cancer cells.
Figure 6 is a diagram showing the tumor growth curve over time in the SKOV3 transplant mouse model after administration of fusion proteins (D310, D311) according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when it is said that a part “includes” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are hereby incorporated by reference.

Throughout this specification, the customary one- and three-letter codes for naturally occurring amino acids are used, as well as the generally accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (Nmethylglycine), etc. Character codes are used. Additionally, amino acids 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 shortens the VH (heavy chain variable) region and VL (light chain variable) region of the antibody. It refers to an antibody in the form of a single chain linked by a peptide chain. The term “scFv” is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or otherwise understood from the context. This is self-evident to those skilled in the art.

The transcription factor CP2c is known to be overexpressed in various cancers, and according to a study by an American research team, inhibiting the expression of CP2c in liver cancer cell lines inhibits growth, and overexpressing CP2c has been reported to cause cancer malignancy and metastasis. [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].

Tumor-targeting antibody fusion protein

In one aspect, the present invention provides a tumor targeting carrier; MMP cleavage site; An anti-cancer peptide and a cell-penetrating peptide are sequentially linked, the tumor-targeting delivery vehicle is an antibody containing an amino acid sequence that specifically binds to a tumor-related antigen, or an antigen-binding fragment thereof, and the MMP cleavage sequence is MMP (Matrix). It relates to a tumor-targeting antibody fusion protein that contains an amino acid sequence that is cleaved by a metalloproteinase.

In one embodiment, the anti-HER2 antibody may be Trastuzumab.

In one embodiment, the antigen-binding fragment may be any one selected from scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2, and preferably may be scFv.

In one embodiment, the tumor targeting delivery vehicle may be represented by any one amino acid sequence selected from SEQ ID NO: 1 to 6, and preferably may be represented by the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the scFv of the anti-HER2 includes a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 39 and a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 40. It may include, and the variable region of the light chain and the variable region of the heavy chain may be connected by a linker containing the amino acid sequence represented by SEQ ID NO: 41.

In one embodiment, the MMP cleavage sequence may be represented by any one amino acid sequence selected from SEQ ID NO: 7 to 10, and preferably may be represented by the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the anticancer peptide may be represented by the amino acid sequence of SEQ ID NO: 11 or 12, and preferably may be represented by the amino acid sequence of SEQ ID NO: 11. The anti-cancer peptide represented by the amino acid sequence of SEQ ID NO: 11 is a CP2c targeting peptide that can bind to the transcription factor CP2c and interfere with the binding of CP2c to DNA.

In one embodiment, the tumor targeting carrier may be one in which a peptide (serum albumin binding peptide (SABP)) containing an amino acid sequence that specifically binds to serum albumin is bound to the N-terminus.

In one embodiment, the SABP may be represented by any one amino acid sequence selected from SEQ ID NO: 13 to 18, and preferably may be represented by the amino acid sequence of SEQ ID NO: 13.

In one embodiment, the anticancer peptide may be a cell penetrating peptide (CPP) containing any one amino acid sequence selected from SEQ ID NOs: 19 to 23 bound to the C-terminus to improve cell penetration, preferably May be a cell-penetrating peptide (iRGD) represented by the amino acid sequence of SEQ ID NO: 19.

In one embodiment, the tumor targeting antibody fusion protein includes albumin binding peptide (saBP); Anti-HER2 or anti-MSLN antibody, or antigen-binding fragment thereof, as a tumor targeting carrier; MMP cleavage sequence; 6xHis to 8xHis; anti-cancer peptides; and cell penetrating peptide; may be sequentially connected.

In one embodiment, the tumor-targeting antibody fusion protein of the present invention is an albumin-binding peptide represented by any one amino acid sequence selected from SEQ ID NOs: 13 to 18; A tumor targeting delivery vehicle represented by any one amino acid sequence selected from SEQ ID NOs: 1 to 6; An MMP cleavage sequence represented by any one amino acid sequence selected from SEQ ID NOs: 7 to 10; Myc His-Tag (6xHis to 8xHis); Anticancer peptide represented by the amino acid sequence of SEQ ID NO: 11 or 12; and a cell-penetrating peptide represented by any one amino acid sequence selected from SEQ ID NOs: 19 to 23.

In one embodiment, the tumor-targeting antibody fusion protein of the present invention may be represented by any one amino acid sequence selected from SEQ ID NOs: 24 to 30.

In the tumor-targeting antibody fusion protein of the present invention, the MMP cleavage sequence is cleaved into a tumor-targeting carrier and an anti-cancer peptide on the outer surface of the cancer cell by the MMP expressed in the cancer cell, and therefore, the tumor-targeting antibody fusion protein, or an antigen-binding fragment thereof, HER2 or other Targeting to tumor-related antigens and targeting cancer cells by anti-cancer peptides can be simultaneously expressed, thereby significantly increasing the ability to kill tumor cells. Accordingly, the tumor-targeting antibody fusion protein of the present invention can significantly improve the low complete response rate seen in existing antibody-based anticancer drugs while maintaining targeting against tumor-related antigens. In addition, the tumor-targeting antibody fusion protein of the present invention can improve the short body half-life of antigen-binding fragment-based anticancer drugs by albumin-binding peptide.

polynucleotide

In another aspect, the present invention relates to a polynucleotide encoding the tumor targeting antibody fusion 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. The polynucleotide of the present invention may be single-stranded DNA or RNA. As used herein, the term “polynucleotide” includes DNA or RNA that contains modified bases and/or unique bases such as inosine. It is obvious that various modifications can be made to the DNA or RNA to serve known useful purposes. As used herein, the term “polynucleotide” 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 tumor targeting antibody fusion protein may be encoded by multiple polynucleotides. In other words, the polynucleotide sequence encoding the tumor-targeting antibody fusion protein available in the present invention is interpreted to include a nucleotide sequence showing substantial identity to any one amino acid sequence selected from SEQ ID NOs: 24 to 30. Preferably, the polynucleotide encoding the tumor-targeting antibody fusion protein of the present invention may include the base sequence represented by SEQ ID NO: 31 or 32.

In one embodiment, the polynucleotide consists of a base sequence represented by SEQ ID NO: 43 and encodes an anti-HER2 scFv; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 44 and encoding an MMP cleavage sequence; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 45 and encoding 8xH; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 46 and encoding a CP2c target peptide; and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 47 and encoding a cell-penetrating peptide (CPP; iRGD).

Preferably, the polynucleotide consists of the base sequence shown in SEQ ID NO: 42 and encodes an albumin-binding peptide; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 43 and encoding anti-HER2 scFv; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 44 and encoding an MMP cleavage sequence; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 45 and encoding 8xH; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 46 and encoding a CP2c target peptide; and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 47 and encoding a cell-penetrating peptide (CPP; iRGD).

In one embodiment, the anti-HER2 scFv may include a polynucleotide (SEQ ID NO: 48) encoding the variable region of the light chain (VL) and a polynucleotide (SEQ ID NO: 49) encoding the variable region (VH) of the heavy chain. and may include a polynucleotide (SEQ ID NO: 50) 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 expressing tumor-targeting antibody fusion proteins

Because scFv is smaller than a regular antibody, it has a high penetration rate into tissue or cancer tissue while maintaining the antigen-specific targeting of the antibody, can be mass-produced in various expression systems including the E. coli system, and can be subjected to various molecular biological modifications. It has the advantage of reducing production time and costs. However, scFv (or tumor-targeting antibody fusion protein containing scFv) has the problem that when expressed in E. coli, it is mostly expressed insoluble, so the amount of functionally usable protein expressed is very small. Accordingly, fusion of the pelB sequence has been attempted to improve the production yield of scFv (or tumor-targeting antibody fusion protein containing scFv) in E. coli, but it was not very effective, and fusion of MBP produces scFv (or scFv). Although it was efficient in solubilizing a tumor-targeting antibody fusion protein containing a fusion protein), an additional purification process was required to separate pure scFv (or a tumor-targeting antibody fusion protein containing an scFv) from the fusion protein, which was cumbersome and time-consuming. Accordingly, the present inventors designed an expression system that can purify soluble scFv (or tumor-targeting antibody fusion protein containing scFv) from E. coli at once without additional processing. According to the expression vector of the present invention and the protein production method using the expression vector, the target protein can be soluble and homogeneously produced in E. coli, and since the target protein is cleaved by the TEV protease expressed within the cell, only the pure target protein is produced. It has the effect of refining in one step.

expression vector

In another aspect, the present invention provides an anti-HER2 or anti-MSLN antibody, or an antigen-binding fragment thereof, as the tumor targeting delivery vehicle; MMP cleavage sequence; anti-cancer peptides; and an expression vector containing a polynucleotide encoding a tumor-targeting antibody fusion protein containing a cell-penetrating peptide.

As used herein, the term “expression” refers to the production of a tumor-targeting antibody fusion protein in cells.

As used herein, the term “expression vector” refers to a vector capable of expressing the tumor-targeting antibody fusion protein in a suitable host cell, and refers to a genetic recombinant containing essential regulatory elements operably linked to express the gene insert. The expression vector refers to a vector containing a nucleic acid sequence encoding at least a portion of the 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. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that also provide other functions.

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

In one embodiment, the polynucleotide may include the base sequence of SEQ ID NO: 31 or 32.

In one embodiment, the expression vector contains a second fusion protein comprising MBP (maltose-binding protein), TEV protease (Tobacco etch virus protease), TEV protease cleavage site, and the tumor-targeting antibody fusion protein. It can manifest.

In one embodiment, the second fusion protein may be sequentially linked to MBP, TEV protease, TEV protease cleavage sequence, and the tumor-targeting antibody fusion protein.

In one embodiment, the MBP may comprise the amino acid sequence of SEQ ID NO: 33, the TEV protease may comprise the amino acid sequence of SEQ ID NO: 35, and the TEV protease cleavage sequence may comprise the amino acid sequence of SEQ ID NO: 37. .

In one embodiment, the second fusion protein may further include 6xHis, 7xHis, or 8xHis.

In one embodiment, the expression vector consists of a base sequence represented by SEQ ID NO: 34, and a polynucleotide encoding MBP; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 36 and encoding TEV protease; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 38 and encoding a TEV protease cleavage sequence; and a polynucleotide consisting of a base sequence represented by SEQ ID NO: 31 or 32 and encoding the tumor-targeting antibody fusion protein.

In one embodiment, the expression vector is capable of solublely expressing a second fusion protein comprising the tumor-targeting antibody fusion protein of the present invention and MBP in E. coli, and the TEV protease in the second fusion protein expressed in the cell is TEV protease. Because the protease cleavage sequence (recognition sequence) is cut (self-cleavage), a pure tumor-targeting antibody fusion protein can be purified in one step.

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

In the present invention, the term “vector” refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). Those skilled in the art can construct vectors by standard recombination 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, expression control sequences such as promoters, terminators, enhancers, etc., depending on the type of host cell for producing the antibody, membrane targeting or secretion Sequences, etc. can be appropriately selected and combined in various ways depending on the purpose.

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

host cell

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

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

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

Method for mass production of protein

In another aspect, the present invention (1) transforms E. coli with an expression vector containing a polynucleotide encoding MBP, TEV protease, a TEV protease cleavage sequence, and a second fusion protein containing the tumor-targeting antibody fusion protein. steps; (2) culturing the E. coli to express a tumor-targeting antibody fusion protein; and (3) purifying the tumor-targeting antibody fusion protein.

In one embodiment, the second fusion protein may be sequentially linked to MBP, TEV protease, TEV protease cleavage sequence, and the tumor-targeting antibody fusion protein.

In one embodiment, the tumor-targeting antibody fusion protein can be purified by Ni-NTA chromatography or a pull-down method using a tag.

In another aspect, the present invention relates to a tumor targeting antibody fusion protein produced by the method of the present invention.

In another aspect, the present invention relates to the use of the tumor-targeting antibody fusion 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 tumor-targeting antibody fusion protein as an active ingredient.

Since the “tumor-targeting antibody fusion protein” of the present invention has already been described in detail, its description is omitted to avoid excessive duplication.

In one embodiment, the cancer may be one or more types selected from breast cancer, ovarian cancer, stomach cancer, rectal cancer, pancreatic cancer, lung cancer, mesothelioma, and endometrial cancer.

The term “prevention” in the present invention means anything that suppresses or delays the cancer.

The term “treatment” as used herein, unless otherwise stated, means reversing, alleviating, inhibiting the progression of, or preventing the symptoms of cancer.

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

The tumor-targeting antibody fusion protein of the present invention can be formulated into a pharmaceutical agent for cancer treatment. The purified protein may be dissolved in a conventional physiologically compatible aqueous buffer solution, to which pharmaceutical excipients may optionally 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 tumor-targeting antibody fusion protein of the present invention may be formulated in lyophilized form or in a stable liquid form. The tumor-targeting antibody fusion protein of the present invention can be lyophilized through various procedures known in the art. Lyophilized formulations are reconstituted prior to use by adding one or more pharmaceutically acceptable diluents, such as sterile water for injection or sterile saline.

The formulation of the composition is delivered to the subject by any pharmaceutically suitable means of administration. A variety of delivery systems are known and may be used to administer the composition by any convenient route. Primarily, the compositions of the invention are administered systemically. For systemic administration, the tumor-targeting antibody fusion protein of the present invention may be delivered parenterally (e.g., intravenously, subcutaneously, intramuscularly, intraperitoneally, intracerebrally, intrapulmonary, intranasally, or transdermally) or enterally (e.g., intravenously) according to conventional methods. Formulated for delivery (oral, vaginal or rectal). The most preferred routes of administration are intravenous and subcutaneous administration. These formulations can be administered continuously by infusion or bolus injection. Some formulations include sustained release systems.

The tumor-targeting antibody fusion protein of the present invention is administered to patients in a therapeutically effective amount, meaning a dose sufficient to produce the desired effect while preventing or reducing the severity or spread of the condition or sign being treated without reaching a dose that causes unacceptable side effects. Administer to The exact dosage depends on various factors such as indication, dosage form, and administration method, and must 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 anti-cancer treatment such as Herceptin.

In one embodiment, the dual-targeting proteins of the present invention are administered in equivalent amounts of about 2, 4, and 12 nanomolar, respectively, and the specific dosage is 0.1 to 50 mg/kg, preferably 0.5 to 30 mg/kg, respectively. More preferably, it may be 1 to 20 mg/kg, and more preferably 3 to 18 mg/kg, respectively. When administration of the dual-targeting 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 through examples, but the present invention is not limited to the following examples.

<Example>

Example 1: Fusion of anticancer peptide and cell penetrating peptide

After various cell-penetrating peptides were fused to the anti-cancer peptide of the present invention, the apoptosis ability of each fusion protein against cancer cells was evaluated, and the results are shown in Figure 2 below.

A cell-penetrating peptide was fused so that the anti-cancer peptide with tumor cell-killing ability can penetrate tumor cells and target intracellular target proteins.

For the above purpose, the anticancer active peptide sequence ACP (SEQ ID NO: 11) or KLAK (SEQ ID NO: 12) and the cell-penetrating peptide sequence iRGD (SEQ ID NO: 19), dNP2 (SEQ ID NO: 20), mdNP2 (SEQ ID NO: 21), mmdNP2 ( The entire amino acid sequence was synthesized by attaching acetyl and amide groups to the N-terminus and C-terminus of cell-penetrating anticancer peptides A to J in the form of SEQ ID NO. 22) or 9R (SEQ ID NO. 23), respectively, and requesting Pepmic Co., Ltd. . The ovarian cancer cell line SKOV-3 was inoculated into a 96-well plate at 4,000 cells/well with 90 μl of culture medium, and then cultured for 4 hours at 37°C and 5% CO2. Add 10 μl of synthetic peptides A to J to each well plate, treat once at concentrations of 0, 0.15, 0.31, 0.63, 1.3, 2.5, 5, and 10 μM, and then culture for 36 hours at 37°C and 5% CO2. did. 10 μl of CCK8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-( 2,4- disulfophenyl)-2H-tetrazolium) solution was added to the well plate after completion of incubation at 37°C. The reaction was performed for 2 hours under 5% CO2 conditions. When the reaction was completed, the absorbance was measured using a microplate reader.

Combinations of specific anti-cancer peptides and cell-penetrating peptides are shown in Table 1 below.

division anti-cancer peptides cell penetrating peptide A ACP
(SEQ ID NO: 11) iRGD (SEQ ID NO: 19) B dNP2 (SEQ ID NO: 20) C mdNP2 (SEQ ID NO: 21) D mmdNP2 (SEQ ID NO: 22) E 9R (SEQ ID NO: 23) F KLAK
(SEQ ID NO: 12) iRGD (SEQ ID NO: 19) G dNP2 (SEQ ID NO: 20) H mdNP2 (SEQ ID NO: 21) I mmdNP2 (SEQ ID NO: 22) J 9R (SEQ ID NO: 23)

Figure 2a is a result of testing the apoptotic ability of the fusion protein against cancer cells, and Figure 2b is a graph showing the quantification of the apoptotic ability of the fusion protein against cancer cells.

Looking at FIG. 2, the anticancer peptide ACP has excellent apoptosis ability against cancer cells when fused with iRGD (“A”), and the anticancer peptide KLAK has excellent apoptosis ability against cancer cells when fused with dNP2, mdNP2, mmdNP2 and 9R (“G”, “H”, “I”, “J”) It can be confirmed that the cell death ability against cancer cells is excellent.

Example 2: Fusion of tumor targeting carrier, anticancer peptide and cell penetrating peptide (scFv-ACP-iRGD)

After preparing a fusion protein by sequentially linking an anti-cancer peptide and a cell-penetrating peptide to the tumor-targeting delivery vehicle of the present invention, we attempted to confirm the binding affinity of the fusion protein to the target antigen and its ability to kill cancer cells.

2-1: Preparation of fusion protein combining tumor-targeting carrier, anticancer peptide, and cell-penetrating peptide

A fusion protein was prepared by sequentially linking an anti-cancer peptide and a cell-penetrating peptide to a tumor-targeting carrier (Figure 3a).

In Figure 3a, the specific sequences of the components constituting each fusion protein are shown in Table 2 below.

division amino acid sequence MBP SEQ ID NO: 33 TEV protease SEQ ID NO: 35 TEV protease cleavage sequence SEQ ID NO: 37 scFv SEQ ID NO: 1 Myc His-Tag (8xH) “HHHHHHHH” ACP SEQ ID NO: 11 iRGD SEQ ID NO: 19

In Table 2, “scFv” refers to the linker sequence (SEQ ID NO: 41) of the light chain variable region (SEQ ID NO: 39) and the heavy chain variable region (SEQ ID NO: 40) of an anti-HER2 antibody (Trastuzumab) to target HER2. It was recombined by linking (SEQ ID NO: 1).

Specifically, the amino acid sequence corresponding to the [TEV cut sequence-scFv-ACP-iRGD] structure of the fusion protein was identified, and the nucleic acid sequence encoding the amino acid sequence was optimized to a codon suitable for expression in E. coli to determine the nucleic acid sequence. .

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

2-2: Expression and purification of fusion protein

In the synthesized nucleic acid sequence, ACP and iRGD were deleted through [MBP-TEV cut sequence-scFv], [MBP-TEV cut sequence-scFv-ACP], and [MBP-TEV cut sequence-scFv-ACP-iRGD] recombinant expression vectors were prepared, respectively.

The nucleic acid sequence of TEV protease was PCR-processed from pRK793 by adding restriction sites SacI site and This was subcloned into the SacI site of the recombinant expression vector to [MBP-TEV-TEV cut sequence-scFv], [MBP-TEV-TEV cut sequence-scFv-ACP], and [MBP-TEV-TEV cut sequence-scFv]. A vector for expressing a fusion protein with the structure of [-ACP-iRGD] was prepared.

The above-prepared fusion protein 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 to select superior strains. Expression of the selected superior strain was again induced in 500 mL of LB liquid medium (Ampicillin 100ug/mL) under the conditions of O.D.600=1.0, IPTG 0.2mM, and 15°C for 16 hr, and about 3 g of bacterial cells were obtained from the culture medium. . Afterwards, the obtained bacterial cells were suspended in PBS to disrupt the cells, and the supernatant thereof was applied to a Ni-NTA column, the column was washed with 25 mM imidazole, and each fusion protein was eluted with 500 mM imidazole. E. coli transformed with the vector for expressing the fusion protein was [MBP-TEV-TEV cut sequence-scFv], [MBP-TEV-TEV cut sequence-scFv-ACP], and [MBP-TEV-TEV cut sequence-scFv- A fusion protein of the [ACP-iRGD] structure was expressed, which was cleaved in E. coli and only [scFv], [scFv-ACP], and [scFv-ACP-iRGD] were purified, respectively.

Briefly, it encodes a fusion protein with the structure of [MBP-TEV-TEV cutting sequence-scFv], [MBP-TEV-TEV cutting sequence-scFv-ACP], and [MBP-TEV-TEV cutting sequence-scFv-ACP-iRGD] structure. Each plasmid was expressed in E. coli (with/without IPTG) and purified by Ni-NTA chromatography using Ni-NTA resin, and the purified proteins were confirmed using SDS-PAGE (FIG. 3b).

Looking at Figure 3b, [scFv], [scFv-ACP], and [scFv-ACP-iRGD] cleaved intracellularly by TEV protease were present in the soluble fraction (S), and were detected by Ni-NTA chromatography or pull-down. It was found to be pure and purified in one go using this method.

On the other hand, when a plasmid encoding a protein prepared by attaching only His6 to the scFv without the MBP-TEV -TEV cleavage sequence is expressed in E. coli, the protein is mostly expressed insoluble and exists in the pellet (P), and is very It was found that only a small amount was expressed soluble.

2-3: Confirmation of binding affinity of fusion protein to target antigen

The purpose was to confirm the binding affinity of the fusion proteins scFv, scFv-ACP, and scFv-ACP-iRGD expressed and purified in 2-2 above to target antigens (HER2 and SKOV3).

Specifically, HER2 (Human Epidermal growth factor receptor2) protein (Acro biosystem, HE2-H5225) was diluted to 50 ng/mL in coating buffer (200mM Na ₂ Co ₃ , 200mM NaHCo ₃ , pH 9.6) and added to 100 ul. Each was dispensed into a 96-well ELISA plate and adsorbed at 4°C overnight. Afterwards, unadsorbed antigen was removed by washing three times with PBS-T (PBS, 0.05% Tween20), and 200 μl of 10% skim milk (PBS, 5% w/v skim milk powder) was added to each well at room temperature. was treated, blocked for 1 hour, and then removed. 100 ul of scFv, scFv-ACP, and scFv-ACP-iRGD expressed and purified in 2-2 above were treated in each well at a concentration of 0.1 pM to 400 nM 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 μl each was treated at room temperature and incubated for 1 hour. Unbound ProteinL-HRP was removed by washing three times with PBS-T (PBS, 0.05% Tween20), and each well was treated with 100 μl of TMB containing 1.5% H ₂ O ₂ at room temperature for 30 minutes. A color reaction was induced. Afterwards, 100 μl of 1M HCL was added to each well to stop the reaction, and the absorbance was measured at 450 nm. Each experiment was repeated three times, and the measured values and standard deviation at each concentration were plotted using the Prism8 program. Additionally, the specific binding affinity (KD value) was determined using ELISA and least-squares fit (LSF) (Figure 3c, left).

In addition, the ovarian cancer cell line SKOV-3 (HER2 overexpressing cell line) cultured in a culture dish at 37°C in a 5% CO ₂ environment was washed and resuspended in 1% BSA (PBS, 1% Bovine Serum Albumin), 10 ⁶ each, 96 cells. It was dispensed into a well U-type bottom plate. Afterwards, 100 μl of the scFv, scFv-ACP, and scFv-ACP-iRGD expressed and purified in 2-2 were added to each well at a concentration of 0.1 pM to 400 nM and incubated at 4°C for 30 minutes. 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). Afterwards, ProteinL FITC (Acrobiosystems, RPL-PF141) was diluted to 5 ug/mL in 1% BSA (PBS, 1% Bovine Serum Albumin), and 100 ul was added to each well and incubated in a dark room at 4°C for 30 minutes. Incubation was carried out. 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. Afterwards, flow cytometry data were derived using a FACSCalibur flow cytometer (BD, USA), and the measured values and standard deviation were plotted using the Prism8 program. Additionally, specific binding affinity (KD value) was determined using flow cytometry and least-squares fit (LSF). (Figure 3c, right).

Looking at Figure 3c, the fusion protein (scFv-ACP-iRGD) prepared by sequentially linking an anti-cancer peptide and a cell-penetrating peptide to a tumor-targeting carrier is a tumor-targeting carrier alone (scFv), or an anti-cancer peptide is connected to the tumor-targeting carrier. It can be seen that the binding affinity to the target antigens (HER2 and SKOV3) is higher than that of the fusion protein (scFv-ACP) prepared.

2-4: Confirmation of apoptosis ability of fusion protein against cancer cells

We sought to confirm the cancer cell killing effects of scFv, scFv-ACP, and scFv-ACP-iRGD using SKOV3 (ovarian cancer cell line) and MDA-MB-231 (breast cancer cell line).

The ovarian cancer cell line SKOV-3 (HER2-overexpressing cell line) was inoculated into a 96-well plate at 4,000 cells/well with 90 μl of culture medium, and then cultured for 4 hours at 37°C and 5% CO2. Then, 10 μl of scFv, scFv-ACP, and scFv-ACP-iRGD expressed and purified in 2-2 above were added to each well plate at concentrations of 0, 0.15, 0.31, 0.63, 1.3, 2.5, 5, and 10 μM, respectively. After treatment once, the cells were cultured for 36 hours at 37°C and 5% CO2 conditions. After the culture was completed, 10 μl of CCK8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) solution was added to the well plate and incubated at 37°C. The reaction was performed for 2 hours under 5% CO2 conditions. When the reaction was completed, the absorbance was measured using a microplate reader.

Looking at Figure 3d, it can be seen that scFv, scFv-ACP, and scFv-ACP-iRGD of the present invention do not exhibit apoptosis ability against cancer cells.

These results infer that when only the tumor-targeting delivery vehicle (scFv) and the anti-cancer peptide are fused without the MMP cleavage sequence of the present invention, the cell death ability can rapidly decrease due to internalization and digestion in the endosome. can do.

Example 3: Fusion of tumor targeting delivery vehicle, MMP cleavage sequence, anticancer peptide and cell penetrating peptide (scFv-MMP-ACP-iRGD)

A fusion protein was prepared by sequentially linking an MMP cleavage sequence, an anti-cancer peptide, and a cell-penetrating peptide to the tumor-targeting delivery vehicle of the present invention, and then the apoptotic ability of the fusion protein against cancer cells was tested.

3-1: Comparison of the apoptosis ability of fusion proteins against cancer cells according to the type of MMP cleavage sequence.

A fusion protein was prepared by sequentially linking the MMP cleavage sequence (MA18, MA15), anticancer peptide, and cell-penetrating peptide to the tumor-targeting carrier (see 2-1 above).

The specific sequences of the components constituting each fusion protein are shown in Table 3 below.

division tumor targeting carrier MMP cleavage sequence anti-cancer peptides cell penetrating peptide D303 SEQ ID NO: 1 (scFv) SEQ ID NO: 8 (MA18) SEQ ID NO: 11 (ACP) SEQ ID NO: 19 (iRGD) D305 SEQ ID NO: 7 (MA15)

After expressing and purifying the fusion protein prepared above, it was confirmed through SDS-PAGE (see 2-2 above) (FIG. 4a).

Afterwards, the apoptotic ability of the fusion proteins (D303 and D305) prepared by varying the MMP cleavage sequences was tested on cancer cells (see 2-4 above) (FIG. 4b).

Looking at Figure 4b, it can be seen that both fusion proteins D303 and D305 prepared with different MMP cleavage sequences exhibit excellent apoptosis ability against cancer cells (SKOV3 and MDA-MB-231).

3-2: Comparison of cell death ability against cancer cells of fusion proteins prepared with different anti-cancer peptides and cell-penetrating peptides

A fusion protein was prepared by sequentially linking the MMP cleavage sequence, anticancer peptide (ACP or KLAK), and cell-penetrating peptide (mmdNP2, 9R, iRGD) to the tumor targeting carrier (see 2-1 above).

The specific sequences of the components constituting each fusion protein are shown in Table 4 below.

division tumor targeting carrier MMP cleavage sequence anticancer peptides cell penetrating peptide D301 SEQ ID NO: 1 (scFv) SEQ ID NO: 8 (MA18) SEQ ID NO: 12 (KLAK) SEQ ID NO: 22 (mmdNP2) D302 SEQ ID NO: 12 (KLAK) SEQ ID NO: 23 (9R) D303 SEQ ID NO: 11 (ACP) SEQ ID NO: 19 (iRGD) D304 - -

After expressing and purifying the prepared fusion protein, it was confirmed through SDS-PAGE (see 2-2 above) (FIG. 4c).

Afterwards, the apoptotic ability of fusion proteins (D301, D302, D303, and D304) prepared by using different anticancer peptides (ACP or KLAK) and cell-penetrating peptides (mmdNP2, 9R, and iRGD) against cancer cells was tested (2 above). -4) (Figure 4d).

Looking at Figure 4d, it can be seen that fusion proteins D301 and D303 prepared with different anti-cancer peptides and cell-penetrating peptides exhibit excellent apoptosis ability against cancer cells (SKOV3 and MDA-MB-231).

Example 4: Fusion of serum albumin binding peptide, tumor targeting delivery vehicle, MMP cleavage sequence, anticancer peptide and cell penetrating peptide (SABP-scFv-MMP-ACP-iRGD)

A fusion protein was prepared by sequentially linking serum albumin-binding peptide, tumor-targeting delivery vehicle, MMP cleavage sequence, anti-cancer peptide, and cell-penetrating peptide while varying the serum albumin-binding peptide.

4-1: Selection of serum albumin binding peptides

The specific sequences of the serum albumin binding peptides used in the experiments of the present invention are shown in Table 5 below.

division amino acid sequence SA20 Ac-QRLIEDICLPRWGCLWEDDF-NH ₂ SA21 Ac-RLIEDICLPRWGCLWEDD-NH ₂ SA22 Ac-RLIEDICLPRWGCLWED-NH ₂ SA29 Ac-RLIEDICLPRWGCLWE-NH ₂ SA31 Ac-RLIEDICLPRWGCLW-NH ₂ SA33 Ac-RLIEDICLPRWGCL-NH ₂ SA35 Ac-RLIEDICLPRWGC-NH ₂ SA23 Ac-LIEDICLPRWGCLWED-NH ₂ SA24 Ac-IEDICLPRWGCLWED-NH ₂ SA25 Ac-EDICLPRWGCLWED-NH ₂ SA26 Ac-DICLPRWGCLWED-NH ₂ SA27 Ac-ICLPRWGCLWED-NH ₂ SA28 Ac-CLPRWGCLWED-NH ₂ SA30 Ac-IEDICLPRWGCLWE-NH ₂

Through research on phage display screening of albumin-binding peptides (Mark S Dennis et. al, Albumin binding as a general strategy for improving the pharmacokinetics of proteins, J Biol Chem. 2002 Sep 20;277(38):35035-43.) Sequences with excellent half maximum binding concentration (IC50) presented in the literature were selected and sequences that specifically bind to albumin were identified.

In addition, when applied to the dual-target anticancer drug platform, the possibility of developing expected hydrophilicity was predicted and compared by expressing it as a hydrophilicity score, and the candidate sequences with excellent predicted values are summarized in Table 6.

division IC50(nM) Hydrophilicity Score SA20 260 0.05 SA21 270±110 0.18 SA22 430±170 0.01 SA29 400±90 -0.17 SA31 200 -0.39 SA33 4,310±2,770 -0.17 SA35 250,000 -0.05 SA23 360±140 -0.18 SA24 1,380±410 -0.07 SA25 2,730±1,300 0.06 SA26 3,120±660 -0.17 SA27 86,700±21,800 -0.43 SA28 400,000 -0.31 SA30 1,800±590 -0.29

From Table 6 above, serum albumin-binding peptides with IC50 of 500 or more were selected and used in the following tests. The specific sequences of the selected serum albumin-binding peptides are shown in Table 7 below.

division sequence number SA20 SEQ ID NO: 13 SA21 SEQ ID NO: 14 SA22 SEQ ID NO: 15 SA29 SEQ ID NO: 16 SA31 SEQ ID NO: 17 SA23 SEQ ID NO: 18

4-2: Preparation of fusion proteins with different serum albumin binding peptides

A fusion protein was prepared by sequentially linking serum albumin-binding peptide, tumor-targeting delivery vehicle, MMP cleavage sequence, anti-cancer peptide, and cell-penetrating peptide while varying the serum albumin-binding peptide (see 2-1 above).

The specific sequences of the components constituting each fusion protein are shown in Table 8 below.

division Serum albumin binding peptide tumor targeting carrier MMP cleavage sequence anti-cancer peptides cell penetrating peptide D310 - SEQ ID NO: 1 (scFv) SEQ ID NO: 8
(MA18) SEQ ID NO: 11
(ACP) SEQ ID NO: 19
(iRGD) D311 SEQ ID NO: 13 (SA20) D312 SEQ ID NO: 14 (SA21) D313 SEQ ID NO: 15 (SA22) D314 SEQ ID NO: 16 (SA29) D315 SEQ ID NO: 17 (SA31) D316 SEQ ID NO: 18 (SA23)

After expressing and purifying the prepared fusion protein, it was confirmed through SDS-PAGE (see 2-2 above) (FIGS. 5A and 5B).

Looking at Figures 5a and 5b, it can be seen that the expression and purification rates of fusion proteins (D310, D311, D312, D314) fused with SA20, SA21, and SA29 are superior.

4-3: Binding affinity of saBP-bound fusion protein to target antigen

The binding affinity of the fusion protein fused with SA20, SA21, and SA29 expressed and purified in 4-2 above to the target antigens (HER2 and SKOV3) was confirmed (see 2-3 above) (FIG. 5c).

Looking at Figure 5c, the fusion proteins (D310, D311, D312, D314) prepared by sequentially linking a tumor-targeting carrier, an anticancer peptide, and a cell-penetrating peptide to saBP have binding affinity for the target antigen and serum albumin. You can see that it is excellent.

4-4: Confirmation of apoptosis ability of fusion protein combining saBP against cancer cells

The apoptotic ability of the fusion protein fused with SA20, SA21, and SA29 expressed and purified in 4-2 above was confirmed for cancer cells (see 2-4 above) (FIG. 5d).

Looking at Figure 5d, the fusion proteins (D310, D311, D312, D314) prepared by sequentially linking a tumor-targeting carrier, an anti-cancer peptide, and a cell-penetrating peptide to the saBP of the present invention all exhibit excellent apoptosis ability against cancer cells. , it can be seen that the fusion of saBP does not inhibit the apoptotic ability of the fusion protein against cancer cells.

Example 5: Tumor growth inhibition effect in xenograft model

Female Balb/c nude mice were 6 weeks old and purchased from KOSABIO, Republic of Korea. Afterwards, they were randomly accepted regardless of experimental group. Mice were raised under controlled laboratory conditions (temperature 22 ± 2 °C, humidity 55 ± 5%, 12-hour light/dark cycle) with sufficient standard feed (Purina Rodent Chow 38057, Republic of Korea) and sterile water. SKOV3 cells were prepared by culturing in a culture dish at 37°C in a 5% CO ₂ environment, and 1×10 ⁷ cells per animal were mixed with 100 μL serum-free medium and injected subcutaneously. Afterwards, when the tumor volume reached 96.6-250.4 mm (width, length, height), the tumors were randomly grouped, and the anticancer candidate proteins Sample D310 and D311 and the reference drug Herceptin were injected iv, respectively. In each experimental group, the sample and control drug were injected at doses of 0, 3, 6, and 18 mg/kg, respectively (0mg/kg, 0.9% Saline with 0.02% Tween20, an injection vehicle (vehicle)). Injection was performed a total of 8 times, once every two days, and the tumors were reared while observing their size. During the injection and observation period, the size of the tumor was measured twice a week for 3 to 4 days, and the measured values and standard deviation of each group of experimental animals were plotted using the Prism8 program. No deaths or abnormal signs were observed in any subjects, including the control group, during the experiment.

Looking at Figure 6, it can be seen that the proteins (D310 and D311) of the examples have a significantly higher tumor growth inhibition effect than the control group (Vehicle). In particular, fusion protein D311, when administered at 6 mg/kg and 18 mg/kg, was found to have a significantly higher tumor growth inhibition effect than the positive control Herceptin (6 mg/kg).

These results mean that the tumor-targeting antibody fusion protein containing the tumor-targeting delivery vehicle (anti-HER2 scFv or anti-MSLN scFv), MMP cleavage sequence, and anticancer peptide of the present invention can be usefully used as a new anticancer therapeutic agent.

In the tumor-targeting antibody fusion protein of the present invention, the anticancer peptide and cell-penetrating peptide have excellent killing ability against tumor cells, and the antibody or antigen-binding fragment thereof has excellent targeting against tumors. The inventors of the present invention have derived a dual-target anticancer drug platform with optimal efficiency by doubling the selective killing ability for tumor cells and increasing half-life in the body by fusing them with the MMP cleavage sequence and albumin-binding peptide (saBP).

Although the present invention has been described in terms of the above-mentioned preferred embodiments, various modifications and variations can be made without departing from the gist and scope of the invention. Additionally, the appended claims cover such modifications or variations as fall within the subject matter of the present invention.

Sequence list electronic file attached

Claims (22)

tumor targeting carrier; MMP cleavage site; An anti-cancer peptide and a cell-penetrating peptide are linked sequentially.
The tumor targeting carrier is an antibody or antigen-binding fragment thereof containing an amino acid sequence that specifically binds to HER2, MSLN or other tumor-related antigen,
A tumor-targeting antibody fusion protein, wherein the MMP cleavage sequence includes an amino acid sequence cleaved by MMP (Matrix metalloproteinase). According to paragraph 1,
The anti-HER2 antibody is Trastuzumab,
A tumor targeting antibody fusion protein, wherein the anti-MSLN antibody is one selected from Amatuximab and Anetumab. According to paragraph 1,
A tumor-targeting antibody fusion protein, wherein the antigen-binding fragment is any one selected from scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2. According to paragraph 1,
The tumor-targeting delivery vehicle is a tumor-targeting antibody fusion protein, characterized in that it is represented by any one amino acid sequence selected from SEQ ID NO: 1 to 6. According to paragraph 1,
A tumor-targeting antibody fusion protein, wherein the MMP cleavage sequence is represented by any one amino acid sequence selected from SEQ ID NOs: 7 to 10. According to paragraph 1,
The anti-cancer peptide is a tumor-targeting antibody fusion protein, characterized in that it is represented by the amino acid sequence of SEQ ID NO: 11 or 12. According to paragraph 1,
The tumor-targeting delivery vehicle is a tumor-targeting antibody fusion protein, characterized in that a peptide (serum albumin binding peptide (SABP)) containing an amino acid sequence that specifically binds to serum albumin is bound to the N-terminus. In clause 7,
The SABP is a tumor-targeting antibody fusion protein, characterized in that it is represented by any one amino acid sequence selected from SEQ ID NOs: 13 to 18. According to paragraph 1,
The anti-cancer peptide is a tumor-targeting antibody fusion protein, characterized in that a cell-penetrating peptide (CPP) containing any one amino acid sequence selected from SEQ ID NOs: 19 to 23 is bound to the C-terminus. An expression vector comprising a polynucleotide encoding the tumor-targeting antibody fusion protein of any one of claims 1 to 9. According to clause 10,
The polynucleotide is an expression vector characterized in that it consists of the base sequence of SEQ ID NO: 31 or 32. According to clause 10,
The expression vector is characterized by expressing a second fusion protein comprising maltose-binding protein (MBP), tobacco etch virus protease (TEV protease), TEV protease cleavage site, and the tumor-targeting antibody fusion protein. expression vector. According to clause 12,
The second fusion protein is an expression vector characterized in that MBP, TEV protease, TEV protease cleavage sequence, and tumor targeting antibody fusion protein are sequentially linked. According to clause 12,
The MBP is an expression vector characterized in that it contains the amino acid sequence represented by SEQ ID NO: 33. According to clause 12,
The TEV protease is an expression vector characterized in that it contains the amino acid sequence represented by SEQ ID NO: 35. According to clause 12,
An expression vector wherein the TEV protease cleavage sequence includes the amino acid sequence represented by SEQ ID NO: 37. The method of claim 12, wherein the expression vector is:
A polynucleotide consisting of the base sequence shown in SEQ ID NO: 34 and encoding MBP;
A polynucleotide consisting of the base sequence shown in SEQ ID NO: 36 and encoding TEV protease;
A polynucleotide consisting of the base sequence shown in SEQ ID NO: 38 and encoding a TEV protease cleavage sequence; and
An expression vector comprising a polynucleotide consisting of a base sequence represented by SEQ ID NO: 31 or 32 and encoding the tumor-targeting antibody fusion protein. A host cell transformed with the expression vector of claim 12. According to clause 18,
The host cell is characterized in that the host cell is E. coli. (1) An expression vector containing a polynucleotide encoding a second fusion protein containing MBP, TEV protease, TEV protease cleavage sequence, and the tumor-targeting antibody fusion protein of any one of claims 1 to 9 was introduced into E. coli. Transforming;
(2) culturing the E. coli to express the tumor-targeting antibody fusion protein; and
(3) Purifying the tumor-targeting antibody fusion protein. A method for mass production of a tumor-targeting antibody fusion protein. A pharmaceutical composition for preventing or treating cancer comprising the tumor-targeting antibody fusion protein of any one of claims 1 to 9 as an active ingredient. According to clause 21,
A pharmaceutical composition, wherein the cancer is at least one selected from breast cancer, ovarian cancer, stomach cancer, rectal cancer, pancreatic cancer, lung cancer, mesothelioma, and endometrial cancer.