KR101347833B1 - CD7-BPB Capable of Binding Specifically to CD7 - Google Patents

Abstract

The present invention relates to bipodal peptide binders (BPBs) that specifically bind to CD7 and uses thereof. The CD7-BPB of the present invention exhibits very low levels (eg, nM levels) of K _d values, resulting in very high affinity to the target. The CD7-BPB of the present invention can be very useful for targeting cells expressing CD7. The CD7-BPB of the present invention, in combination with other structures (preferably liposomes), allows for the delivery of the desired drug to cells expressing CD7 (preferably T cells).

Description

CD7-BPB Capable of Binding Specifically to CD7}

The present invention relates to CD7-BPB and its use for specifically binding to CD7.

Cluster of Differentiation 7 (CD7) is a 40 kDa protein found primarily in hematopoietic stem cells. CD7 is expressed on the surface of mature T cells and natural killer cells (NK cells) (Aruffo, Embo J. 6: 3313 (1987); Yoshikawa, Immunogenetics 33: 352 (1991); Yoshikawa, Immunogenetics 37: 114 (1993) Yoshikawa, Immunogenetics 41: 159 (1995)). Although not all functions of the CD7 protein in the immune system are known, signaling via CD7 is known (Carrera, J. Immunol 141: 1919 (1988); Rabinowich, J. Immunol Vol. 153: 3504 (1994)).

CD7 is known to play important functions in T cell interactions and T-cell / B-cell interactions.

CD7 deficient mice have been reported to be completely resistant to low levels of LPS-induced shock and partially resistant to high levels of LPS-induced shock, suggesting that CD7 is involved in the LPS-induced shock pathway. (Sempowski et al., J. Exp. Med. 189, 1011-1016 (1999)).

In addition, Dr. Lee et al. Successfully targeted CD7 targeting T cell-specific siRNA to inhibit HIV-1 infection in humanized mice (Cell, Vol 134, 577-586, 22 August 2008).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

We sought to develop peptide molecules that specifically bind human CD7 proteins. As a result, we developed a CD7 specific BPB using the basic structure of the bipodal peptide binder (BPB) previously developed by the present inventors, and this CD7 specific BPB has a very high affinity for CD7 and thus CD7 surface expression. The present invention was completed by proving that the targeting ability to the cells is very excellent.

Accordingly, an object of the present invention is to provide a CD7 specific bipodal peptide binder (BPB).

Another object of the present invention is to provide a liposome in which a CD7 specific bipodal peptide binder (BPB) is bound to a surface.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a CD7 specific bipodal peptide binder (BPB) having an amino acid sequence of SEQ ID NO: 1.

We sought to develop peptide molecules that specifically bind human CD7 proteins. As a result, we developed a CD7 specific BPB using the basic structure of the bipodal peptide binder (BPB) previously developed by the present inventors, and this CD7 specific BPB has a very high affinity for CD7 and thus CD7 surface expression. It was found that the targeting ability to the cells was very good.

The present invention basically adopts the structure of the BPB. BPB is an affinity peptide originally proposed by the present inventors, and a detailed description thereof can be found in WO 2010/047515.

As used herein, the term " peptide " refers to a linear molecule formed by peptide bonds and amino acid residues joined together.

Peptides of the invention can be prepared according to chemical synthesis methods known in the art, in particular solid-phase synthesis techniques (Merrifield, J. Amer . Chem . Soc . 85: 2149-54 (1963) Stewart, et al., Solid Phase Peptide Synthesis , 2nd. ed., Pierce Chem. Co .: Rockford, 111 (1984)).

The CD7 specific BPB of the present invention has a very high affinity for CD7. As demonstrated in the examples below, the CD7 specific BPBs of the invention exhibit an equilibrium binding constant (K _d ) value of about 93 nM for CD7. Through this excellent affinity, the CD7 specific BPB of the present invention can be very useful for targeting cells expressing CD7.

According to another aspect of the present invention, the present invention provides a liposome in which the CD7 specific bipodal peptide binder (BPB) is bound to the surface.

CD7 specific BPBs in liposomes of the invention serve as targeting ligands.

The term " liposome " as used herein refers to a lipid carrier made by forming a closed lipid bilayer. Liposomes are lipid bilayer membranes that are biocompatible and biocompatible, allowing them to pass through hydrophobic membranes with internal hydrophilic materials.

As used herein, the term “phospholipid” means a phospholipid capable of forming liposomes, unless otherwise specified, and refers to lecithin (phosphatidyl choline), phosphatidyl serine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin Including but not limited to. Lecithin can be obtained from eggs, soybeans and other plants, most preferably soybean lecithin. Synthetic, semi-synthetic and natural lecithins that can be used in the present invention include soybean lecithin, distearoylphosphatidylcholine, hydrogenated soybean lecithin, egg lecithin, dioleoylphosphatidylcholine, hydrogenated egg lecithin, dielaidoylphosphatidylcholine, diuretic Palmitoylphosphatidylcholine and dimyristoylphosphatidylcholine.

According to a preferred embodiment of the present invention, the liposome of the present invention is encapsulated with a chemical drug, protein, peptide or nucleotide molecule as a cargo therein.

As used herein, the term “encapsulation” refers to encapsulation to enclose a delivery material and to efficiently incorporate it in vivo.

Proteins or peptides carried by the liposomes of the present invention are not particularly limited, and include hormones, hormone analogs, enzymes, inhibitors, signaling proteins or portions thereof, antibodies or portions thereof, single chain antibodies, binding proteins or binding domains thereof, Antigens, adhesion proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulators, blood coagulation factors and vaccines, and the like. More specifically, the protein or peptide carried by the drug carrier of the present invention is insulin, insulin-like growth factor 1 (IGF-1), growth hormone, erythropoietin, granulocyte-colony stimulating factors ), GM-CSFs (granulocyte / macrophage-colony stimulating factors), interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin-3, interleukin-4, interleukin-6, interleukin-2, EGFs (epidermal growth factors, calcitonin, adrenocorticotropic hormone (ACTH), tumor necrosis factor (TNF), atobisban, buserelin, cetrorelix, deslorelin , Desmopressin, dynorphin A (1-13), elcatonin, eleidosin, eptifibatide, growth hormone releasing (GHRH-II) hormone-II), gonadorelin, goserelin, hisstrin, Leuprorelin, lypressin, octreotide, octootide, oxytocin, pitressin, secretin, sincalide, terlipressin, Thymopentin, thymosine α1, triptorelin, bivalirudin, carbetocin, cyclosporin, exedine, lanreotide, Luteinizing hormone-releasing hormone (LHRH), nafarelin, parathyroid hormone, pramlintide, T-20 (enfuvirtide), thymalfasin and ziconotide.

The chemicals carried by the liposomes of the present invention are not particularly limited and include, for example, acibaicin, aclarubicin, acodazole, acronycin, adozelesin, alanosine, aldehydes, allopurinol Sodium, Althetamine, Aminoglutetimide, Amonaphide, Ampligen, Amsacrine, Androgens, Anguidine, Apidicholine glycinate, Asaray, Asparaginase, 5-Azacytidine, Aza Thioprine, Bacillus Calmette-Guerin (BCG), Bakers Antipol, Beta-2-Dioxythioguanosine, Bisanthrene HCl, Bleomycin Sulfate, Bullhorn, Butionine Suloximine, BWA 773U82, BW 502U83 / HCl, BW 7U85 mesylate, cerasemide, carbetamer, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladry Wien, corticosteroids, corinbacterium Reboom, CPT-11, Crisnatol, Cyclocytidine, Cyclophosphamide, Cytarabine, Cytembena, Davies maleate, Decarbazine, Dactinomycin, Daunorubicin HCl, Diazauridine, Dexarasonic acid , Dihydrohydrogalactitol, diajicuone, dibromodulcitol, dididemin B, diethyldithiocarbamate, diclicoaldehyde, dihydro-5-azacytin, doxorubicin, echinomycin, deda trek Sate, edelfosine, epronitine, elliots solution, elsamutrucin, epirubicin, esorubicin, estramastine phosphate, estrogen, ethandiazole, ethiophos, etoposide, padrazole, pazarabine, Fenretinide, filgrastim, finasteride, flavone acetic acid, phloxuridine, fludarabine phosphate, 5-fluorouracil, Fluosol ™, flutamide, gallium nitrate, gemcitabine, goserelin a Tate, Hepsulfam, Hexamethylene Bisacetamide, Homohartontonin, Hydrazine Sulfate, 4-hydroxyandrostenedione, Hydroziurea, Idarubicin HCl, Iphosphamide, 4-Ipomeanol, Iproplatin, Isotretinoin, Leucovorin Calcium, Lyuprolide Acetate, Levamisol, Romerstin, Rodinamine, Maytansine, Mechloretamine Hydrochloride, Melphalan, Menogaryl, Merbaron, 6-mercaptopurin, Mesna, Bacillus cal Methanol extract of Lete-Guerin, methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin-C, mitotan, mitoxantrone hydrochloride, monosite / macrophage colony-stimulating factor, nabilone, Napoxidine, neocarcinostatin, octreotide acetate, ormaplatin, oxaliplatin, paclitaxel, pala, pentostatin, piperazindione, blood Pobromann, pyrarubicin, pyritrexim, pyroxanthrone hydrochloride, PIXY-321, plymycin, porpimer sodium, prednisostin, procarbazine, progestins, pyrazopurin, lazoic acid, sargramos Tim, semustine, spirogermanium, spiromostin, streptonaigreen, streptozosin, sulofener, suramin sodium, tamoxifen, taxorere, tegapur, teniposide, terephthalamidine, theroxyron, thioguanine, thio Tefa, thymidine injection, thiazopurin, topotecan, toremifene, tretinoin, trifluoroperazine hydrochloride, trifluuridine, trimetrexate, uracil mustard, vinblastine sulfate, vincristine sulfate, vindesine, vino Lelvin, vinzolidine, Yoshi 864, and zorubicin.

Nucleotide molecules carried by liposomes of the present invention include antisense oligonucleotides, siRNAs, shRNAs, miRNAs, aptamers, ribozyme and DNAzyme.

As used herein, the term “antisense oligonucleotide” refers to DNA or RNA or derivatives thereof that contain a nucleotide sequence complementary to a sequence of a particular mRNA, which binds to a complementary sequence within the mRNA and translates the mRNA into a protein. There is a characteristic that inhibits. The antisense nucleotide sequence of the present invention refers to a DNA or RNA sequence complementary to the mRNA of the target gene and capable of binding to the mRNA of the target gene, and is capable of translating, mRNA, And may inhibit essential activities for all other overall biological functions. The length of the antisense oligonucleotide is 6 to 100 bases, preferably 8 to 60 bases, more preferably 40 bases in 10.

The antisense oligonucleotides can be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol . , 5 (3): 343-55, 1995). Oligonucleotide backbones can be modified with phosphorothioates, phosphoroesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic intersaccharide bonds, and the like. In addition, antisense oligonucleotides may include one or more substituted sugar moieties. Antisense oligonucleotides may comprise modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-me pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this.

The term “aptamer” refers to an oligonucleotide (generally an RNA molecule) that binds to a specific target. Preferably, “aptamer” in the context of the present invention means oligonucleotide aptamer (eg, RNA aptamer). For general information about aptamers, see Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med . 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).

The term "siRNA" refers to a short double-chain RNA that can induce RNA interference (RNAi) through the cleavage of a particular mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. siRNA is provided as an efficient gene knock-down method or gene therapy because it can inhibit the expression of the target gene.

siRNAs are not limited to the complete pairing of double-stranded RNA pairs paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), etc. May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, most preferably 20 to 70 bases. The siRNA terminal structure can be blunt or cohesive, as long as the expression of the target gene can be suppressed by the RNAi effect. The cohesive end structure is possible in both a three-terminal protruding structure and a five-terminal protruding structure. The number of protruding bases is not limited. For example, the base water may be from 1 to 8 bases, preferably from 2 to 6 bases. In the present specification, the total length of the siRNA is represented by the sum of the length of the central double-stranded portion and the length of the single-stranded protrusion at both ends. In addition, siRNA is a low-molecular RNA (e.g., a natural RNA molecule such as tRNA, rRNA, viral RNA or artificial RNA) in the protruding portion of one end to the extent that can maintain the expression inhibitory effect of the target gene Molecules). The siRNA terminal structure does not need to have a cleavage structure on both sides, and a stem loop structure in which one terminal region of double-stranded RNA is connected by linker RNA may be used. The length of the linker is not particularly limited as long as it does not interfere with the pairing of the stem portions. The siRNA of the present invention has an antisense RNA strand comprising a sequence complementary to a target gene or a target nucleotide so as to specifically act on a target gene.

The term “shRNA” refers to 50-70 nucleotides in single strands, in Stem-loop structure in vivo . Long RNAs of 19-29 nucleotides complementary to both loop regions of 5-10 nucleotides form base pairs to form a double stranded stem.

The term “miRNA (microRNA)” regulates gene expression and refers to a single stranded RNA molecule consisting of 21-23 nucleotides in length. miRNAs are oligonucleotides that are not expressed intracellularly and have a short stem-loop structure. miRNAs are homologous, in whole or in part, with one or more mRNAs (messenger RNAs) and inhibit target gene expression through complementary binding to the mRNAs.

The term “ribozyme” is a type of RNA that has the same function as an enzyme that recognizes and cleaves itself on the base sequence of a particular RNA. Ribozyme is a complementary nucleotide sequence of a target messenger RNA strand consisting of a region that binds with specificity and a region that cleaves the target RNA.

The term “DNAzyme” is a single-stranded DNA molecule with enzymatic activity. DNAzyme (10-23 DNAzyme) consisting of 10-23 nucleotides cleaves RNA strands at specific positions under physiologically similar conditions. 10-23 DNAzymes do not form a base pair and cleave between any purines and pyrimidines that are exposed. 10-23 DNAzyme (DNAzyme) is an active site of the enzyme consisting of 15 conserved sequences (5'-GGCTAGCTACAACGA-3 ') and 7- to recognize RNA substrates to the left and right of the enzyme. It consists of an RNA substrate binding domain consisting of eight DNA sequences.

According to a preferred embodiment of the invention, the liposome of the present invention is encapsulated therein a complex comprising (i) a nucleotide molecule as a cargo and (ii) a cationic molecule. Liposomes characterized by.

Cationic molecules used as complexes in the present invention include cationic peptides, cationic polymers and cationic natural proteins (eg, protamine).

The cationic peptide is preferably composed of human immunodeficiency virus (HIV) -Tat, 6-40 amino acid residues, at least three of which are lysine, arginine or lysine and arginine peptides, KALA or protamine, more preferably Preferably consisting of 8-15 amino acid residues and at least three of said amino acid residues are lysine, arginine or a peptide that is lysine and arginine, even more preferably 8-10 amino acid residues and at least 6 of said amino acid residues. Dogs are peptides that are lysine, arginine or lysine and arginine, most preferably arginine peptides consisting of 9 arginine residues.

According to a preferred embodiment of the present invention, the liposome of the present invention has a ratio of charge (negative charge: positive charge) of the nucleotide molecule as a cargo and the cationic peptide as the cell penetration peptide is 1: 1-12. More preferably 1: 4-9, and most preferably 1: 5-7. If the charge ratio is exceeded, there is a problem that the transfer efficiency of the gene is reduced.

According to another preferred embodiment of the invention, the liposomes of the present invention have an average size of 80-200 nm, more preferably 90-150 nm, most preferably 100-120 nm.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides CD7-BPB that specifically binds to CD7.

(Ii) The CD7-BPB of the present invention exhibits very low levels (eg, nM levels) of K _d values, resulting in very high affinity to the target.

(Iii) The CD7-BPB of the present invention can be very useful for targeting cells expressing CD7.

(iv) The CD7-BPB of the present invention, in combination with other structures (preferably liposomes), is intended to deliver the desired drug to cells that express CD7 (preferably T cells).

1 is a diagram showing the schematic (a) and colony screening results for the bipodal peptide binder (BPB) library.
Figure 2 is a diagram showing the results of PCR for cloning the human CD7 protein.
3 is a diagram showing the results of CD7-Trx expression induction.
4 is a diagram showing the results of CD7-Trx treatment with thrombin to separate CD7 and Trx by gel filtration to obtain a CD7 protein.
5 is a biopanning schematic for selecting phages that specifically bind to the extracellular domain of human CD7.
6 is a graph showing the biopanning results of human CD7 BPB.
7 is BPB1 _CD7 This is a graph confirming the specificity of phage human CD7.
Figure 8 BPB1 _CD7 against human _CD7 It is a graph measuring the affinity of phage.
9 is BPB1 _CD7 The binding affinity of peptides is analyzed by FACS.
10 is a schematic diagram of a BPB-conjugated nanocarrier.
11 is a schematic of the construction of siRNA-encapsulated BPB1 _CD7 -conjugated liposome nanocarriers.
12 is a diagram showing the intake uptake efficiency of BPB1 _CD7- LS.
FIG. 13 shows an in-bit uptake efficiency of BPB1 _CD7- LS by FACS.
14 is a graph confirming the in vitro CD4 siRNA knockdown efficiency.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1 Construction of Bipodal Peptide Binder (BPB) Library

To construct a library of BPBs (FIG. 1A), two oligonucleotides Beta-F1 (5'-TTCTATGCGGCCCAGCTGGCC (NNK) ₆ GGATCTTGGACATGGGAAAACGGAAAA-3 ') and Beta-B1 (5'-AACAGTTTCTGCGGCCGCTCCTCC TCC (MNN) ₆ TCCCTTCGGT-3 ') (N is A, T, G or C; K is G or T; M is C or A). To make a double chain, 50 μl of 4 μM of Beta-F1, 4 μM of Beta-B1, 4 μl of 2.5 mM dNTP mixture, 1 μl of ExTaq DNA polymerase (Takara, Seoul, Korea) and 5 μl of 10 × PCR buffer A total of 25 mixtures with distilled water added were prepared. This mixture was subjected to PCR reaction (5 minutes at 94 ° C, 60 cycles: 30 seconds at 30 ° C, 30 seconds at 72 ° C and 7 minutes at 72 ° C) to make a double chain, followed by PCR purification kit (GeneAll, Seoul, Korea) Purification was performed to obtain a bipodal peptide binder gene. In order to connect the gene to be inserted into the bipodal peptide binder to the pIGT2 phagemid vector (Ig therapy, Chuncheon, Korea), the restriction gene was treated to the insert gene and the pIGT2 phagemid vector. About 11 μg of insert DNA was reacted with SfiI (New England Biolabs (NEB, Ipswich) and NotI (NEB, Ipswich) for 4 hours and purified using a PCR purification kit. Also, about 40 μg of pIGT2 phagemid The vector was reacted with SfiI and NotI for 4 hours, and then added to CIAP (Calf Intestinal Alkaline Phosphatase) (NEB, Ipswich) for 1 hour, and then purified using a PCR purification kit. Quantified by Ultrospec 2100pro, Amersham Bioscience, 2.9 μg of insert gene was linked with 12 μg of pIGT2 phagemid vector at 18 ° C. using T4 DNA ligase (Bioneer, Daejeon, Korea) for 15 hours, and then precipitated with ethanol. TE buffer to dissolve the DNA in 100 ㎕. Competence for ligation results consultant E. coli A library of 8 x 10 ⁸ BPB clones was obtained by electroporation into XL1-BLUE cells (American Type Culture Collection, Manassas, USA). The transformed cells were infected with 1 × 10 ¹¹ pfu of Ex helper phage (Ig therapy, Chuncheon, Korea) to amplify the BPB-displayed phage library.

To evaluate the library, PCR colony screening and DNA sequencing were performed. PCR colony screening revealed that the 16/16 random picking clones obtained from the library contained insert sequences of the correct size of 126 bp (FIG. 1B). Sequencing of 20 clones revealed that 50% inserts were in frame.

Example 2: Cloning and Purification of Human CD7 Proteins

Nucleotide sequences for the extracellular domain of human CD7 (residues 26-176) were obtained by PCR, and sense primers 5′-AAA GGA TCC GCC CAA GAG GTG CAG-3 ′ and antisense primers 5′-ATT CTC GAG TCA GGC TGC TGG CGG GTC AGG-3 ′ was used. The amplification product (FIG. 2) was cleaved with BamHI / XhoI, ligated to a pET32 expression vector (thioredoxin (Trx) fusion vector) and transformed into E. coli strain C41 (DE3) (FIG. 2). ).

The transformed E. coli 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to C41 (DE3) and incubated at 37 ° C. for 10 hours to induce the expression of human CD7-Trx (FIG. 3). The bacteria were then collected, resuspended and sonically crushed, and the lysates were centrifuged. The supernatant was collected and loaded into a column packed with Ni-NTA affinity resin, then the column was washed and His-tag fused CD7-Trx protein was eluted. Fractions containing CD7-Trx and Trx were collected and purified by gel filtration using a Superdex 200 column. 3 mg of CD7-Trx was obtained from the CD7-Trx fraction. To obtain CD7 protein, CD7-Trx was treated with thrombin and CD7 and Trx were separated by gel filtration (FIG. 4).

Example 3: Biopanning and Phage ELISA for Screening of CD7-Specific BPB

In order to select the phage that specifically binds to the extracellular domain of human CD7, general biopanning was performed (FIG. 5). 96-well ELISA plates (Maxisorb) were coated with 1 μg of CD7-Trx and Trx proteins, respectively, and blocked with 2% BSA. Trx was used for counting. In the first round of biopanning, 1.0 × 10 ¹¹ cfu phage libraries were added to CD7-Trx coated wells and incubated at 37 ° C. for 2 hours to allow binding. Unbound phage was removed with 0.1% PBST and then bound phage was eluted with 0.2 M glycine-HCl (pH 2.2). All fractions of the phage were titered to assess the degree of selection during biopanning.

To screen for BPBs that specifically bind to recombinant human CD7 (rhCD7), a BPB library consisting of 8 x 10 ⁸ independent clones was used for s-tag-rhCD7 bound to an anti-s-tag antibody immobilization plate. Biopanning was performed. Anti-s-tag antibodies were counted for all rounds of biopanning to reduce non-target binding phage. RhCD7 binding phage was recovered by performing excessive target elution with 25 μg / ml rhCD7 in rounds 1 and 2. After round 3, bound phages were eluted with acidic buffer (0.2 M glycine-HCl, pH 2.2). After the fourth round of biopanning, significant enrichment (250-fold increase) of rhCD7 BPB was achieved (FIG. 6). After 4 rounds, rhCD7 binding capacity of 60 random selection clones was evaluated by ELISA, resulting in 10 positive phages. As a result of DNA sequencing, BPB1 _CD7 ASTINFGSWTWENGKWTWKGYTRRWN that specifically binds to the extracellular domain of human _CD7 was obtained.

BPB1 _CD7 To analyze phage clones, rhCD7, anti-s-tag antibody, human tumor necrosis factor alpha (hTNF), streptavidin, bovine serum albumin (BSA) and humans at a concentration of 1 x 10 ⁹ cfu / ml Phage ELISA was performed on VEGF. Experimental results, BPB1 _CD7 Phage showed a very high specificity for rhCD7 (FIG. 7).

Example  4: SPR ( Surface Plasmon Resonance Affinity measurement by

To determine the affinity of BPB1 _CD7 for rhCD7, BPB1 _CD7 Peptides were synthesized (Anigen, Korea). Affinity measurements were performed using BIAcore X (Biacore AB, Uppsala, Sweden). PBS (pH 7.4) was used as the ning buffer, and flow was flowed at 30 μl / min and the kinetics were measured at various concentrations. The affinity was calculated using BIAevaluation software (Biacore AB, Uppsala, Sweden).

Experimental results show that BPB1 _CD7 exhibits 1.4 x 10 ⁵ M ^-1 s ^-1 binding rate constant (k _a ) and 1.3 x 10 ^-2 s ^-1 dissociation rate constant (k _d ), resulting in an equilibrium binding constant of about 93 nM ( K _d ) value can be seen (FIG. 8).

Example  5: FACS  analysis

BPB1 _CD7 The binding affinity of the peptides was analyzed by fluorescence-activated cell sorting (FACS). CD7 positive and negative cells were incubated with 1 μg FITC-BPB1 _CD7 peptide at 4 ° C. for 20 minutes and the cells washed three times by centrifugation. At least 100,000 cells per sample were evaluated. As shown in FIG. 9, CD7 positive cell lines (human Jurkat T cells, human Molt4 T cells) showed significant binding of 92% and 99%, respectively. However, CD7 negative cell lines (mouse NS1 B cells, mouse Raw macrophages, human U87MG glioma) showed very weak binding of 16%, 3% and 5%, respectively. These experimental results show that the BPB1 _CD7 peptide is specific for CD7.

Example  6: BPB Construction of Junction Nanocarriers

Liposome-based nanocarriers were selected as model nanocarriers in order to construct nanocarriers with high loading and excellent stability and human affinity.

A. Conjugation of BPB to Liposomal Nanocarriers

Before preparing whole liposomes, BPB1 _CD7 should be conjugated to phospholipids. Cysteine modified BPB1 _CD7 500 was reacted with 1 mg of Mal-PEG2000-DSPE (1: 2 ratio) overnight with stirring (FIG. 10). Success of conjugation was confirmed by MALDI-TOF. The conjugate was purified by HPLC (using YMC C4 column).

B. siRNA Enclosed Construction of bonded nano liposome carriers - BPB1 _CD7

siRNA was complexed with 9R (peptide consisting of 9 Arg residues) prior to siRNA inclusion. The siRNA sequences used were as follows: sense strand, 5'-GAU CAA GAG ACU CCU CAG U (dTdT); Antisense strand, 5'-ACU GAG GAG UCU CUU GAU C (dTdT).

Liposomes were prepared with a combination of POPC / Chol / POPG / (molar ratio 4: 3: 3). PEG2000-DSPE was used for long term circulation in vivo. BPB1 _CD7- PEG2000-DSPE (1% of total molar ratio) was added to the flask and the chloroform was dried under vacuum and further dried by lyophilizer. 9R / siRNA complexes in 5% HBG buffer were added to the dried lipid film and allowed to form liposomes. To prepare liposomes of uniform size, liposomes were injected through a 100 nm polycarbonate membrane (FIG. 11).

Example  7: BPB1 _CD7 - LS Rated in beat of

A. BPB1 _CD7 - in vitro uptake efficiency of the LS

In vitro uptake efficiency of BPB1 _CD7 -LS (liposomes constructed in Example 6 above) was analyzed by confocal microscopy and FACS. LS (liposome) and BPB1 _CD7 -LS particles were fluorescently stained with rhodamine-1,2-distearolyl- sn -glycerero-3-phosphoethanolamine. Human Jurkat T cells and mouse monocytes RAW cells were used as positive and negative cells, respectively. As adherent cells, RAW cells were grown on gelatin coated coverslips because of anchorage, and Jurkat T cells were grown on normal Petri dishes. Both cells were treated with non-targeting liposomes (LS) and BPB1 _CD7 -LS at 200 total lipid concentrations, and the cells were fixed at 3 and 6 hour time points (for RAW cells) and observed under confocal microscopy. As can be seen in FIG. 12A, BPB1 _CD7 -LS was uptaken intracellularly in human Jurkat T cells expressing CD7 and BPB1 _CD7 -LS was not uptakeed intracellularly in RAW cells not expressing _CD7 (FIG. 12B). ).

Subsequently, FACS analysis was performed to quantify the amount of nanoparticles incorporated into Jurkat T cells. Jurkat T cells were seeded at 1 × 10 ⁵ cell density per well. Cells were treated at 200 total lipid concentrations with rhodamine labeled non-targeting liposomes (LS) and BPB1 _CD7 -LS. FACS analysis was performed at the 3 and 6 hour time points.

As can be seen in Figure 13, it can be seen that a significant amount of BPB1 _CD7 -LS has been incorporated into Jurkat T cells.

B. CD4 In Bit siRNA knockdown efficiency

CD4 siRNA / 9R complexes were encapsulated in nontargeting liposomes and BPB1 _CD7- LS at a concentration of 1 / ml. For the knockdown test, Jurkat T cells were grown to 80% confluency in 6-well plates. Cells were treated with 100 nM lipofectamine / siRNA complex (positive control), LS (siRNA / 9R) and BPB1 _CD7 -LS (siRNA / 9R). All experiments were repeated 3 times. After 4 hours treatment in serum-free medium, cells were washed three times with PBS and then incubated for 24 hours. Cells were then collected and subjected to real time RT-PCR to analyze CD4 mRNA levels. As can be seen in Figure 14, knockdown of CD4 was highest in cells treated with BPB1 _CD7 -LS (siRNA / 9R).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Attach an electronic file to a sequence list

Claims (8)

delete A liposome having a CD7 specific bipodal peptide binder (BPB) having an amino acid sequence of SEQ ID NO: 1 listed on the surface thereof.
The liposome of claim 2, wherein the liposome targets a cell expressing CD7.
The liposome according to claim 2, wherein the liposome is encapsulated with a chemical drug, a protein, a peptide, or a nucleotide molecule as a cargo therein.
The liposome according to claim 4, wherein the liposome is encapsulated with a complex containing (i) a nucleotide molecule as a cargo and (ii) a cationic molecule.
6. The method of claim 5 wherein the cationic molecule is a cationic peptide and the cationic peptide consists of human immunodeficiency virus (HIV) -Tat, 6-40 amino acid residues and at least three of the amino acid residues are lysine, arginine or Liposomes, characterized in that selected from the group consisting of lysine and arginine peptides, KALA and protamine.
The double-stranded ODN of claim 4, wherein the nucleotide molecule of the carrier is an antisense oligonucleotide, aptamer, siRNA, shRNA, miRNA, ribozyme, DNAzyme, or transcription factor binding sequence. Liposomes).
4. The liposome according to claim 3, wherein the cell expressing CD7 is a T cell.

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