KR20240161932A - The immunotherapeutic nanocage displaying LAG3 binding peptide and use in anti-cancer immunotherapeutic agent thereof - Google Patents

Abstract

The present invention relates to a ferritin nanocage fused with a LAG3 binding peptide and its use as an anticancer immunotherapeutic agent. Peptides (LAG3pep1: CIRNDPAVC and LAG3pep2: CSVLNASGC) that bind to the immune checkpoint receptor LAG3 are each fused to a protruding loop on the surface of human ferritin to form a cage, thereby producing a nanocage in which 24 LAG3 binding peptides are displayed. The nanocage of the present invention specifically binds to the LAG3 receptor and blocks LAG3, thereby enhancing immune activity, and effectively targets brain cancer tissues by passing through the BBB to exhibit an anticancer effect. In addition, an immune checkpoint blocking nanocage fused with a LAG3 binding peptide and a PD-L1 binding peptide specifically binds to each receptor and exhibits a dual-functional immune checkpoint blocking effect. Therefore, the nanocage technology of the present invention is expected to be a next-generation drug that overcomes the limitations of immune checkpoint blocking therapy using antibodies.

Description

{The immunotherapeutic nanocage displaying LAG3 binding peptide and use in anti-cancer immunotherapeutic agent thereof}

The present invention relates to a ferritin nanocage fused with a LAG3 binding peptide and its use as an anticancer immunotherapy agent, and more particularly, to the use of the ferritin nanocage fused with the LAG3 binding peptide for immune checkpoint control and combined anticancer immunotherapy.

Cage proteins are proteins that can form macromolecules tens to hundreds of times the molecular weight of a single molecule by precise self-assembly of low-molecular-weight single molecules. In nature, viral capsid proteins, ferritin, heat shock proteins, and Dps proteins are examples of this, and each single molecule that composes the cage interacts very regularly and precisely with adjacent single molecules, and the interior of the cage is an empty structure. Due to the container-like properties of the cage protein as described above, it has the characteristic of isolating the interior and the exterior, and is frequently utilized as a drug delivery vehicle in the medical field.

In the field of cage protein application material transport, research on viral vectors and non-viral vectors is being actively conducted. Adenoviruses and other viral transporters have been studied extensively so far, and ferritin and heat shock proteins have been studied as non-viral transporters. However, in the case of existing viral transporters, safety issues have been raised due to the genes possessed by the virus itself.

Ferritin protein is a type of intracellular protein that stores and releases iron. Ferritin generally has a hollow spherical cage shape in the body, and the cage is composed of 24 subunits, and the subunits are divided into heavy chains and light chains according to their structure.

Recently, selective monoclonal antibodies targeting immune checkpoint molecules have achieved unprecedented success in the treatment of human cancers. This development of cancer immunotherapy marks a new milestone in the history of cancer treatment. Lymphocyte-activation gene 3 (LAG3), one of the immune checkpoint molecules, is an attractive therapeutic target in several cancers. It is mainly upregulated upon T cell activation and is also abundant in exhausted T cells commonly found in tumor-infiltrating lymphocytes. Panel studies of human and mouse tumor samples have shown that LAG3 is highly expressed in lymphocytes in secondary immune organs as well as in tumor-infiltrating lymphocytes. LAG3 is expressed in various types of lymphocytes and dendritic cells.

Although a significant number of cancer patients respond to blockade therapy of major immune checkpoints such as CTLA-4, PD-1, and PD-L1, many patients still do not show a response. In addition, when immune cells (T-cells) that cause anticancer effects are exposed to cancer tissues, they express other immune checkpoint inhibitor proteins such as LAG3 in addition to PD-1, thereby losing the anticancer effect, and immune checkpoint inhibitory therapy that blocks PD-1/PD-L1 does not appear in effect. Therefore, there is a need to develop a drug delivery platform that can enhance the immune checkpoint inhibitory function and stability of peptides that bind to LAG3 and can effectively transport them to cancer tissues.

Korean Patent Publication No. 10-2022-0053848 (Published on May 2, 2022)

The present invention provides a fusion polypeptide having a LAG3 binding peptide inserted into a human ferritin heavy chain fragment consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, a fusion polypeptide further having a PD-L1 binding peptide consisting of an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 bound to the N-terminus of the fusion polypeptide, and a ferritin nanocage comprising the fusion polypeptide.

In addition, the present invention provides a polynucleotide encoding the fusion polypeptide, an expression vector comprising the polynucleotide, and an isolated transformant transformed with the expression vector.

In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating cancer containing the ferritin nanocage as an active ingredient.

In addition, the present invention aims to provide a drug delivery composition comprising the ferritin nanocage as an active ingredient.

To solve the above problem, the present invention provides a fusion polypeptide having a LAG3 binding peptide inserted into a human ferritin heavy chain fragment consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, a fusion polypeptide further having a PD-L1 binding peptide consisting of an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 bound to the N-terminus of the fusion polypeptide, and a ferritin nanocage comprising the fusion polypeptide.

Additionally, the present invention provides a polynucleotide encoding the fusion polypeptide.

In addition, the present invention provides an expression vector comprising the polynucleotide.

In addition, the present invention provides an isolated transformant transformed with the expression vector.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer containing the ferritin nanocage as an active ingredient.

In addition, the present invention aims to provide a drug delivery composition comprising the ferritin nanocage as an active ingredient.

The present invention relates to a ferritin nanocage fused with a LAG3 binding peptide and its use as an anticancer immunotherapeutic agent. Peptides (LAG3pep1: CIRNDPAVC and LAG3pep2: CSVLNASGC) that bind to the immune checkpoint receptor LAG3 are each fused to a protruding loop on the surface of human ferritin to form a cage, thereby producing a nanocage in which 24 LAG3 binding peptides are displayed. The nanocage of the present invention specifically binds to the LAG3 receptor and blocks LAG3, thereby enhancing immune activity, and effectively targets brain cancer tissues by passing through the BBB to exhibit an anticancer effect. In addition, an immune checkpoint blocking nanocage fused with a LAG3 binding peptide and a PD-L1 binding peptide specifically binds to each receptor and exhibits a dual-functional immune checkpoint blocking effect. Therefore, the nanocage technology of the present invention is expected to be a next-generation drug that overcomes the limitations of immune checkpoint blocking therapy using antibodies.

Figure 1 shows the design and purification results of immune checkpoint proteins fused with Lag3 binding peptide 1 (CIRNDPAVC) or 2 (CSVLNASGC).
Figure 2 shows the results of comparative analysis of the binding affinity of each manufactured fusion protein (cultured cell binding and protein binding analysis using a surface plasmon resonance device).
Figure 3 shows the results of designing a bifunctional PDL1/Lag3 blocking ferritin fusion protein by simultaneously displaying PDL1 binding peptide 1 or 2 or Lag3 binding peptide 1 or 2 on ferritin.
Figure 4 shows the results of observing the cage formation and particle size of each manufactured fusion protein using TEM and DLS.
Figure 5 shows the results of comparative analysis of binding affinity (cultured cell binding) of each manufactured fusion protein.
Figure 6 shows the tumor-targeting effect of the immune checkpoint blocking nanocage.
Figure 7 shows the results of the in vivo immune anticancer effects of P1L1 and P1L2.
Figure 8 shows the results of biotoxicity confirmation of Lag3 binding peptide display ferritin fusion proteins (L1, L2, P1L1, P1L2) through blood and serum analysis.

The present invention provides a fusion polypeptide in which a LAG3 binding peptide is inserted into a human ferritin heavy chain fragment, comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

In addition, the present invention provides a fusion polypeptide in which a PD-L1 binding peptide consisting of an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 is additionally linked to the N-terminus of the fusion polypeptide.

Preferably, the fusion polypeptide may be one in which the LAG3 binding peptide is inserted into the loop region of the 4th and 5th helices of the human ferritin heavy chain fragment.

Preferably, the LAG3 binding peptide may be, but is not limited to, SEQ ID NO: 3 or SEQ ID NO: 4.

Preferably, the portion additionally bonded in the fusion polypeptide may further include a linker.

Meanwhile, the fusion polypeptides P1L1, P1L2, P2L1 and P2L2 described in the present invention may each be composed of an amino acid sequence represented by SEQ ID NO: 7 to SEQ ID NO: 10.

In addition, the present invention provides a ferritin nanocage comprising the fusion polypeptide. Specifically, the ferritin nanocage may be in a form in which a LAG3 binding peptide and a PD-L1 binding peptide are fused to the outer surface of ferritin.

In the present invention, ‘ferritin’ is a type of intracellular protein that stores and releases iron. Ferritin generally has a hollow spherical cage shape in a living body, and the cage is composed of 24 ferritin monomers, and the ferritin monomers are classified into heavy chains and light chains according to their structure. In the present invention, the ferritin protein may be used without limitation as long as each of them is an active protein that can form a cage-shaped complex protein as a unit.

In the present invention, a "nanocage" is a protein cage formed by the precise self-assembly property of low-molecular-weight monomers and having a space inside. Virus capsid proteins, ferritin, heat shock proteins, and Dps proteins are examples of these. The nanocage of the present invention is characterized by containing the fusion polypeptide of the present invention as a monomer constituting the nanocage. The term "self-assembly" used in the present invention means a property where certain molecules form a specific nanostructure by themselves without any special external stimulation or artificial induction.

The nanocage of the present invention may be a complex protein in which the fusion polypeptide of the present invention is regularly arranged as a unit, and more preferably, it may be formed by regularly arranging 24 fusion polypeptides of the present invention in a three-dimensional manner.

Additionally, the present invention provides a polynucleotide encoding the fusion polypeptide.

The above “polynucleotide” is a polymer of deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form. It includes RNA genome sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogues of natural polynucleotides unless specifically stated otherwise.

The above polynucleotide comprises not only a nucleotide sequence encoding the above fusion polypeptide, but also a complementary sequence thereto. The complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence.

In addition, the polynucleotide may be modified. The modification includes addition, deletion, or non-conservative or conservative substitution of nucleotides. The polynucleotide encoding the amino acid sequence is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. The substantial identity may be a sequence that exhibits at least 80% homology, at least 90% homology, or at least 95% homology when the nucleotide sequence and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

In addition, the present invention provides an expression vector comprising the polynucleotide.

In addition, the present invention provides an isolated transformant transformed with the expression vector.

In the present invention, “vector” means a self-replicating DNA molecule used to transport a clone gene (or other piece of clone DNA).

In the present invention, “vector” means a plasmid, viral vector or other vector known in the art capable of expressing an inserted nucleic acid in a host cell, and may be a polynucleotide encoding the fusion polypeptide of the present invention operably linked to a conventional expression vector known in the art. The expression vector may generally include a replication origin capable of proliferating in a host cell, one or more expression control sequences (e.g., promoter, enhancer, etc.) that control expression, a selective marker, and a polynucleotide encoding the fusion polypeptide of the present invention operably linked to the expression control sequence. The transformant may be one transformed by the expression vector.

Preferably, the transformant can be obtained by introducing an expression vector comprising a polynucleotide encoding the fusion polypeptide of the present invention into a host cell by a method known in the art, including but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun and other known methods for introducing nucleic acids into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer containing the ferritin nanocage as an active ingredient.

Preferably, the cancer may be, but is not limited to, breast cancer, colon cancer, rectal cancer, brain tumor, lung cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, stomach cancer, bladder cancer, ovarian cancer, bile duct cancer, gallbladder cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, and squamous cell carcinoma.

Preferably, the pharmaceutical composition can inhibit immune checkpoints.

The pharmaceutical composition of the present invention can be manufactured using pharmaceutically suitable and physiologically acceptable auxiliary agents in addition to the effective ingredient, and the auxiliary agents can be solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the effective ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and can be used by mixing one or more of saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents as needed. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound, etc. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the age, weight, general health condition, sex and diet of the patient, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, and drugs used simultaneously. Although not limited thereto, for example, in the case of adults, the composition of the present invention may be administered once to several times a day, and in the case of a compound, may be administered at a dose of 0.1 ng/kg to 10 g/kg.

In addition, the present invention provides a drug delivery composition comprising the ferritin nanocage as an active ingredient.

The ferritin nanocage according to the present invention can be used as an intelligent drug delivery vehicle that selectively delivers drugs to cancer tissues. If a conventionally known drug is loaded into the ferritin nanocage of the present invention and used for cancer treatment, the drug is selectively delivered only to cancer tissues and cancer cells by the ferritin nanocage of the present invention, so the efficacy of the drug can be increased and at the same time, the side effects of the drug on normal tissues can be significantly reduced.

The above drug is an anticancer agent, and any anticancer agent that can be loaded into the ferritin nanocage of the present invention can be used without limitation as long as it is conventionally used in the treatment of cancer. Examples include doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, teniposide, bisantrene, homoharringtonine, Gleevec (STI-571), cisplatin, 5-fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, and nitrosoureas.

Hereinafter, the present invention will be described in detail based on examples that do not limit the present invention. It should be noted that the following examples of the present invention are intended only to concretize the present invention and do not limit or restrict the scope of the rights of the present invention. Therefore, it is interpreted that what can be easily inferred by a specialist in the technical field to which the present invention belongs from the detailed description and examples of the present invention falls within the scope of the rights of the present invention.

< Example 1> Design and purification of immune checkpoint proteins fused with Lag3 binding peptide 1 (CIRNDPAVC) or 2 (CSVLNASGC)

1. Experimental method

(1) Production of Lag3N1, Lag3L1, Lag3C1 (cage with Lag3 peptide1 inserted into the N-terminus, loop of helix 4 and 5, and C-terminus) and Lag3N2, Lag3L2, Lag3C2 (cage with Lag3 peptide2 inserted into the N-terminus, loop of helix 4 and 5, and C-terminus) DNA

Human ferritin heavy chain fragment (1-161) gene sequence or human ferritin heavy chain fragment (1-183) gene sequence was amplified by PCR from a cDNA library reverse transcribed from human mRNA and inserted into the pET-28a plasmid using a genetic recombination method. The gene sequence of Lag3-binding peptide 1 or 2 was chemically synthesized and inserted into the nitrogen terminus of human ferritin heavy chain fragment (1-161) using restriction enzymes KpnI and NheI or into the carbon terminus of human ferritin heavy chain fragment (1-161) using restriction enzymes SpeI and XhoI. When inserting the peptide into the nitrogen terminus of ferritin, a GG sequence peptide was inserted in between to increase the degree of freedom, and when inserting the peptide into the carbon terminus of ferritin, a longer flexible linker (GGGSGGGGSG) was inserted to increase the degree of freedom and prevent the peptide from being hidden inside ferritin. Additionally, the gene sequence of Lag3 binding peptide 1 or 2 was inserted between the genes corresponding to the 4th and 5th helices of the human ferritin heavy chain (1-183) gene using restriction enzymes BamHI and ApaI.

(2) Purification of Lag3N1, Lag3L1, Lag3C1 and Lag3N2, Lag3L2, Lag3C2 proteins

E. coli (BL21) transformed with each plasmid was inoculated into LB medium and grown in a 37°C shaking incubator. When the OD ₆₀₀ value became 0.5, IPTG was added to the medium to 1 mM to induce protein overexpression. Lysis buffer was added to E. coli and disrupted by sonication. The lysate (supernatant) collected by centrifugation was reacted with Ni-NTA beads at 4°C for 2 hours, and the lysate that did not bind to the beads was filtered through a chromatography column, and the beads were washed with wash buffer containing 30 mM imidazole. The endotoxin of E. coli was removed with wash buffer containing 0.1% Triton X-114, and Triton X-114 was removed with wash buffer. Proteins were eluted and purified using each elution buffer containing 100 mM and 300 mM imidazole, respectively. For each protein, more than 95% homogeneous purified protein was recovered by SDS PAGE.

2. Experimental Results

The human ferritin heavy chain fragment has three junction points at the N-terminus, a loop connecting helices 4 and 5, and the C-terminus, which enable peptide display on the surface when 24 monomers polymerize to form a cage. In the present invention, fusion proteins (Lag3N1, Lag3L1, Lag3C1 and Lag3N2, Lag3L2, Lag3C2) in which Lag3-binding peptide 1 or 2 was inserted into each of these three junction points were designed and manufactured, and the differences in structure, biophysical properties, and binding affinity were clarified (Fig. 1).

The 3D structural models of Lag3N1, Lag3L1, and Lag3C1 calculated through computer simulation are as shown in Fig. 1A, and the gene sequence schematic is as shown in Fig. 1B. Fig. 1C shows the results of verifying stable expression and purification in E. coli by confirming protein bands at the same positions as the theoretical monomer sizes of each protein by performing electrophoresis of each protein by SDS-polyacrylamide gel electrophoresis and staining the gel with Coomassie Brilliant Blue.

< Example 2> Comparative analysis of the binding strength of each manufactured fusion protein (protein binding analysis using cultured cell binding and surface plasmon resonance device)

1. Experimental method

Confocal laser scanning microscopy: HEK293T cells were transfected with GFP-Lag3 and cells transfected with GFP only (mock), and then treated with Lag3 antibody to confirm the expression of GFP-Lag3. To prevent nonspecific binding of proteins, 1% BSA in DMEM was used to block the cells at room temperature for 30 minutes. After removing the BSA, the fusion proteins of Lag3N1, Lag3L1, Lag3C1 and Lag3N2, Lag3L2, Lag3C2 (100 nM) were reacted at 4℃ for 1 hour. After washing with PBS, the cells were stained (green) by reacting with anti-human ferritin heavy chain antibody (1:500 ratio) for 1 hour. The cells were fixed with 4% paraformaldehyde and observed using a confocal microscope. To observe the nucleus, DAPI was dissolved in PBS at a ratio of 1:1000 and stained (blue) for 5-10 minutes at room temperature (Fig. 2A).

It is known that Lag3 receptor is expressed when Jurkat T cells are treated with phorbol 12-myristate 13-acetate (PMA), Ionomycin, and Chloroquine. After drug treatment for 48 hours, Lag3N1, Lag3L1, Lag3C1 and Lag3N2, Lag3L2, Lag3C2 fusion proteins (100 nM) labeled with FITC fluorescence were reacted at 4°C for 1 hour. After washing with PBS, the binding strength was analyzed by reading the fluorescence of the cells using a flow cytometer. At this time, the labeled fluorescence of each added protein was corrected to have the same value. Binding to cells not treated with IFN-γ was observed as a control (Fig. 2B).

Lag3-Fc protein was coated on the surface of a gold sensor chip for surface plasmon resonance to which protein A was attached. The Lag3N1, Lag3L1, Lag3C1 and Lag3N2, Lag3L2, Lag3C2 fusion proteins dissolved in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM DTT solution were flowed onto the gold sensor chip to which Lag3-Fc protein was attached, respectively, and the binding kinetics were analyzed. The right channel without Lag3-Fc protein was used as a reference to correct the resonance measurement values (Fig. 2C).

2. Experimental Results

Lag3N1, Lag3L1, Lag3C1 and Lag3N2, Lag3L2, Lag3C2 fusion proteins bound to HEK293T cells expressing Lag3. No difference in binding affinity could be observed between each fusion protein. In contrast, when the binding to Jurkat cells expressing Lag3 was quantitatively analyzed by flow cytometry, Lag3L1 and Lag3L2 showed better binding affinity than the other fusion proteins. When the binding affinity to the purified Lag3 receptor was measured using a surface plasmon resonance device, it was confirmed that Lag3L1 and Lag3L2 showed better binding affinity than the other fusion proteins. Based on these results, it was concluded that the Lag3-binding peptide would have excellent binding function when inserted into the loop of the 4th and 5th helices of ferritin (Fig. 2).

< Example 3> PDL1 Combine Peptide 1 or 2 or Lag3 Combine Peptide 1 or 2 to ferritin By displaying them simultaneously Dual functionality PDL1 / Lag3 Blocking Ferritin fusion protein design

Lag3 is expressed in exhausted CD8+T cells and exhibits an immunomodulatory function, and is also expressed in other immune cells such as Treg cells, so when blocked alone, the anticancer modulatory function was found to be minimal. However, it is known that the anticancer effect is doubled when blocked in combination with the PD-L1/PD-1 immune checkpoint regulatory function. In fact, many clinical trials are in progress for the combined administration of antibodies that block PD-L1/PD-1 and Lag3 antibodies. Previous studies have revealed that when a PD-L1 binding peptide is inserted into the N-terminus of ferritin, it exhibits an anticancer effect by binding and blocking PD-L1. Based on this, a bifunctional fusion protein was designed in which PD-L1 binding peptide 1 (CLQKTPKQC) or PD-L1 binding peptide 2 (CVRARTR) was inserted into the N-terminus of ferritin and Lag3 binding peptide 1 or 2 was inserted into the loop of the 4th and 5th helices (Fig. 3).

< Example 4> Each manufactured fusion protein TEM and With DLS Cage Observation of formation and particle size

Each protein was purified using Ni-NTA beads from E. coli overexpressing P1L1, P1L2, P2L1, and P2L2-ICBN (immune checkpoint blocking nanocage) fusion proteins, and transmission electron microscopy (TEM) analysis was performed to confirm whether the fusion proteins formed a ferritin nanocage. It was confirmed that each protein formed a nanocage, and the size of each cage was confirmed by performing dynamic light scattering (DLS) analysis (Fig. 4).

< Example 5> Each manufactured fusion protein Comparative analysis of binding affinity (cultured cells and Surface plasmon resonance device (via combined analysis)

1. Experimental method

Confocal laser scanning microscopy: HEK293T cells transfected with GFP-Lag3 and cells transfected with only GFP (mock) were treated with Lag3 antibody to confirm the expression of GFP-Lag3 in GFP-Lag3-transfected cells. To prevent nonspecific binding of proteins, 1% BSA in DMEM was used to block the cells at room temperature for 30 minutes. After removing the BSA, the cells were reacted with Lag3L1, Lag3L2, P1L1, P1L2, P2L1, P2L2-ICBN, or wild type ferritin (100 nM) at 4℃ for 1 hour. After washing with PBS, the cells were stained with anti-human ferritin heavy chain antibody (1:500 ratio) for 1 hour (green). The cells were fixed with 4% paraformaldehyde and observed using a confocal microscope. To observe the nucleus, DAPI was dissolved in PBS at a ratio of 1:1000 and stained (blue) for 5-10 minutes at room temperature (Fig. 5A).

The binding to triple-negative breast cancer cells (MDA-MB 231) expressing PD-L1 was measured. MCF-7, breast cancer cells that do not express PD-L1, were observed as a control. To prevent nonspecific binding of PD-L1 protein, 1% BSA was dissolved in the media and blocked at room temperature for 30 minutes. Then, BSA was removed and PpNF (P1), Pp2NF (P2), P1L1, P1L2, P2L1, P2L2-ICBN, or wild type ferritin (100 nM) was reacted at 4℃ for 1 hour. After washing with PBS, anti-human ferritin heavy chain antibody (1:500 ratio) was reacted for 1 hour and stained (green). After fixation with 4% paraformaldehyde, the samples were observed under a confocal microscope. To observe the nucleus, DAPI was dissolved in PBS at a ratio of 1:1000 and stained (blue) for 5-10 minutes at room temperature (Fig. 5B).

After coating the Lag3 protein on the surface of a gold sensor chip for surface plasmon resonance, the Lag3L1, Lag3L2, P1L1, and P1L2 ICBN fusion proteins dissolved in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM DTT solution were flowed onto the gold sensor chip bound to the Lag3 protein, and the binding kinetics were analyzed (Fig. 5C).

2. Experimental Results

P1L1, P1L2, P2L1, and P2L2-ICBN fusion proteins bound to HEK293T cells expressing Lag3 and MDA-MB231 cells expressing PD-L1. They did not bind to HEK293T cells (mock transfected) that do not express Lag3, indicating specific binding to Lag3. They did not bind to MCF-7 cells that do not express PD-L1, but nonspecific binding was observed with P2, P2L1, and P2L2. The wt ferritin control group did not bind to any cells. Based on this, it was determined that P1L1 and P1L2 act more specifically on PD-L1 (Fig. 5). When the binding affinity to the purified Lag3 receptor was measured using a surface plasmon resonance device, it was found that Lag3L1, Lag3L2, P1L1, and P1L2 all bound specifically. Among these, P1L2 had the best binding strength.

< Example 6> Immune checkpoints Blocking Nanocage's Tumor targeting effect

1. Experimental method

1× ¹⁰⁶ MC38 colon cancer cells in 100 ㎕ of PBS were injected subcutaneously into the right thigh of 6-week-old BALB/c mice. When the tumor size was approximately 100 ㎣, 200 ㎕ of P1L2 (10 mg/kg) and ferritin wildtype (10 mg/kg) proteins each labeled with Flamma-675 and dissolved in saline were injected into the tail vein of each mouse.

The biodistribution of the protein was investigated at 1, 4, 8, 12, 18, and 24 hours after injection using the IVIS fluorescence imaging system. Then, 48 hours later, the mice were sacrificed and the tumor, heart, lung, liver, kidney, and spleen were removed to investigate how much protein remained in the organs.

In addition, confocal laser scanning microscopy was performed. After culturing glioblastoma cells expressing the PD-L1 receptor (GL26), P1, P2, P1L1, P1L2, P2L1, P2L2 ICBN (100 nM) was reacted at 4℃ for 1 hour. After washing with PBS, the cells were stained with anti-human ferritin heavy chain antibody (1:200 ratio) for 1 hour (green). After fixation with 4% paraformaldehyde, the cells were observed under a confocal microscope. To observe the nucleus, DAPI was diluted in PBS at a ratio of 1:1000 and stained for 5-10 minutes at room temperature (blue). As controls, wild-type ferritin and PpNF (P1) and Pp2NF (P2), which have been verified to bind to PD-L1, were used.

2. Experimental Results

It was found that P1L2 had increased tumor targeting ability compared to wild-type ferritin (tumor targeting by EPR effect) (Fig. 6A).

In addition, we confirmed that the P1L1, P1L2, P2L1, and P2L2 bifunctional fusion proteins bind to glial cells expressing PD-L1 in the same manner as P1 and P2 displaying PD-L1 binding peptides (Fig. 6B). Based on this, we inferred that P1L1 and P1L2 can be targeted and developed as glioblastoma therapeutics, just as PpNF passes through the BBB and is targeted to glioblastoma.

< Example 7> P1L1 , P1L2's Immuno-oncogenic effects in vivo

1. Experimental method

MC38 cells were injected subcutaneously into the upper right thigh of BALB/c wild-type mice. When the tumor size was approximately 100 mm3, the mice were randomly divided into four groups. P1, L1, L2, P1L1, P1L2, or wt Ferritin dissolved in saline, bicarbonate buffer (pH 7.4) at a concentration of 10 mg/kg each, and therapeutic anti-PD-L1 antibody, Lag3 antibody were injected into the tail vein of each mouse. The tumor size and body weight of the mice were measured during the five injections, once every two days (anti-PD-L1 antibody was injected three times, once every three days) (Fig. 7B and Fig. 7C).

2. Experimental Results

P1L1 and P1L2 fusion proteins significantly inhibited tumor growth compared to P1 or L1, L2 fusion proteins (Fig. 7B). In particular, P1L2 showed a superior anticancer effect than the group co-administered with PDL1 and Lag3 antibodies. There was no change in body weight compared to the control mice in all fusion protein administration groups (Fig. 7C).

These results showed that P1L2 effectively inhibits tumor growth through its immune checkpoint suppression effect.

< Example 8> Through blood and serum analysis Lag3 Combine Peptide Display ferritin fusion protein (L1, L2, P1L1 , P1L2 )of Biotoxicity check

After the antitumor animal experiment, when the mice were sacrificed, blood and serum were collected from each group, and the amounts of red blood cells, hemoglobin, hematocrit, white blood cells, and neutrophils in the blood and blood urea nitrogen, creatinine, alanine aminotransferase, and aspartate aminotransferase in the serum were measured and compared.

In the group of mice treated with the Lag3 binding peptide display ferritin fusion protein, no hepatotoxicity or renal toxicity was observed, and there were no significant changes in hematology (Fig. 8).

While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Attach electronic file of sequence list

Claims (14)

A fusion polypeptide comprising a human ferritin heavy chain fragment and a LAG3 binding peptide, the fusion polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. In the first paragraph, the fusion polypeptide is a fusion polypeptide characterized in that the LAG3 binding peptide is inserted into the loop region of the 4th and 5th helices of the human ferritin heavy chain fragment. A fusion polypeptide, characterized in that in claim 1, the LAG3 binding peptide has SEQ ID NO: 3 or SEQ ID NO: 4. A fusion polypeptide further comprising a PD-L1 binding peptide comprising an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6, attached to the N-terminus of the fusion polypeptide according to claim 1. A ferritin nanocage comprising a fusion polypeptide according to any one of claims 1 to 4. In claim 5, the ferritin nanocage is characterized in that the ferritin nanocage is in a form in which a LAG3 binding peptide and a PD-L1 binding peptide are fused to the outer surface of ferritin. A polynucleotide encoding a fusion polypeptide according to any one of claims 1 to 4. An expression vector comprising the polynucleotide of claim 7. A transformant isolated by transformation with the expression vector of Article 8. A pharmaceutical composition for preventing or treating cancer, comprising the ferritin nanocage of claim 5 as an active ingredient. A pharmaceutical composition for preventing or treating cancer, characterized in that in claim 10, the cancer is any one selected from the group consisting of breast cancer, colon cancer, rectal cancer, brain tumor, lung cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, stomach cancer, bladder cancer, ovarian cancer, bile duct cancer, gallbladder cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, and squamous cell carcinoma. In claim 10, the pharmaceutical composition is a pharmaceutical composition for preventing or treating cancer, characterized in that it inhibits an immune checkpoint. A drug delivery composition comprising the ferritin nanocage of claim 5 as an active ingredient. A drug delivery composition, characterized in that the drug in claim 13 is an anticancer agent.