Fusion protein comprising antibody specifically binding to CLDN3 and interferon-beta variant, and combination therapy using same

Abstract

The present invention relates to a fusion protein comprising an antibody specifically binding to CLDN3 and an interferon-beta variant, and a combination therapy using same. By combining an immunocytokine, in which an IFN-β variant is fused to a tumor-targeting CLDN3 antibody, with photothermal therapy (PTT), it is possible to not only eliminate local tumors but also induce a systemic antitumor immune response, and thus the present invention can be usefully employed as a therapeutic strategy for the prevention or treatment of cancer.

Description

A fusion protein comprising an antibody specifically binding to CLDN3 and an interferon-beta variant, and a combination therapy using the same

[0001] The present invention relates to a fusion protein comprising an antibody that specifically binds to CLDN3 for the prevention or treatment of cancer and an interferon-beta variant, and a combination therapy of said fusion protein with photothermal therapy or an immune checkpoint inhibitor.

[0002] Recently, photothermal therapy (PTT) has garnered attention as a treatment method that kills tumor cells by generating localized heat through the irradiation of near-infrared (NIR) lasers onto photothermal substances selectively accumulated in tumor tissue. Furthermore, it has been suggested that PTT may induce the so-called abscopal effect, which exhibits anti-tumor effects on distant tumors by not only eliminating local tumors but also promoting an immune response through the induction of tumor antigen release.

[0003] However, according to current studies, PTT monotherapy alone has limitations in inducing a strong systemic immune response, and it has been reported that combination therapy with immunomodulatory factors, such as immune checkpoint inhibitors or immunostimulatory cytokines, is necessary to induce a sufficient abscopal effect.

[0004] Interferon-beta (IFN-β) is known as a cytokine capable of directly inducing apoptosis in tumor cells, while simultaneously enhancing anti-tumor immune responses through the activation of immune cells. Therefore, immunotherapy using IFN-β is presented as a promising strategy for inducing anti-tumor immune responses in the tumor microenvironment.

[0005] Meanwhile, Claudin-3 (CLDN3) is known to be a cell membrane protein that is overexpressed in various types of cancer, including colorectal cancer, and is being studied as a promising target for tumor-targeted therapy. Accordingly, various studies are being conducted to deliver drugs or functional proteins specifically to tumors using antibodies targeting CLDN3.

[0006] In this regard, a nanoplatform for photothermal therapy using antibody-conjugated polydopamine (PDA) nanoparticles has been developed in prior research (Korean Registered Patent No. 10-2692009), and it has also been reported that an antibody-cytokine fusion protein in which an IFN-β variant is fused to an antibody can induce tumor-specific immune activation (Korean Registered Patent No. 10-2651002).

[0007] However, despite these technologies, therapeutic strategies capable of effectively linking localized photothermal therapy with systemic immune responses to suppress metastatic tumors and prevent recurrence have not yet been sufficiently established.

[0008] Accordingly, the inventors completed the present invention by preparing an antibody-cytokine fusion protein in which an IFN-β variant is fused to a CLDN3 target antibody, and by confirming that this can induce a potent abscopal effect and enhance an anti-tumor immune response by combining it with photothermal therapy and immune checkpoint inhibitors.

[0009] The object of the present invention is to provide an IFN-β variant in which the 17th amino acid of human IFN-β, cysteine, is substituted with serine and the 27th amino acid, arginine, is substituted with threonine; and a fusion protein comprising an anti-claudin 3 antibody or an antigen-binding fragment thereof.

[0010] Another object of the present invention is to provide a polynucleotide encoding the fusion protein, an expression vector comprising the polynucleotide, and a host cell transformed with the expression vector.

[0011] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein as an active ingredient.

[0012] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein as an active ingredient and used in combination with a phospholipid-photothermal nanoparticle complex having an anti-Claudin-3 antibody or an antigen-binding fragment thereof bound to its surface.

[0013] Another objective of the present invention is to provide a method for preventing or treating cancer, comprising the step of administering an effective amount of the fusion protein to a subject.

[0014] Another object of the present invention is to provide a method for preventing or treating cancer comprising the step of co-administering to a subject the fusion protein and a phospholipid-photothermal nanoparticle complex having an anti-Claudin-3 antibody or an antigen-binding fragment thereof bound to its surface.

[0015] In order to achieve the above objective,

[0016] The present invention provides an IFN-β variant in which the 17th amino acid of human IFN-β, cysteine, is substituted with serine and the 27th amino acid, arginine, is substituted with threonine; and a fusion protein comprising an anti-claudin 3 antibody or an antigen-binding fragment thereof.

[0017] The present invention provides a polynucleotide encoding the fusion protein, an expression vector comprising the polynucleotide, and a host cell transformed with the expression vector.

[0018] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein as an active ingredient.

[0019] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein as an active ingredient and used in combination with a phospholipid-photothermal nanoparticle complex having an anti-Claudin-3 antibody or an antigen-binding fragment thereof bound to its surface.

[0020] The present invention provides a method for preventing or treating cancer, comprising the step of administering an effective amount of the fusion protein to a subject.

[0021] The present invention provides a method for preventing or treating cancer, comprising the step of co-administering to a subject the fusion protein and a phospholipid-photothermal nanoparticle complex having an anti-Claudin-3 antibody or an antigen-binding fragment thereof bound to its surface.

[0022] The present invention relates to a fusion protein comprising an antibody that specifically binds to CLDN3 and an interferon beta variant, and a combination therapy using the same. By combining an immunocytokine in which an IFN-β variant is fused to a tumor-targeting CLDN3 antibody with photothermal therapy (PTT), it is possible to induce not only local tumor removal but also a systemic anti-tumor immune response, and thus it can be usefully used as a therapeutic strategy for the prevention or treatment of cancer.

[0023] Figure 1a is a schematic diagram showing the structure of a fusion protein in which an interferon-beta variant is combined with an anti-claudin 3 antibody.

[0024] Figure 1b is a schematic diagram of a phospholipid-photothermal nanoparticle complex with a tumor-targeting antibody bound to its surface.

[0025] Figure 2a shows the results of verifying the in vitro photothermal therapy efficacy of antibody-photonanoparticle conjugates using mouse tumor cells by checking the temperature change during light irradiation.

[0026] Figure 2b shows the results of analyzing the survival rate of mouse tumor cells following photothermal treatment with an antibody-photonanoparticle conjugate.

[0027] Figure 3 shows the results of analyzing immunogenic cell death on the cell surface by applying heat stress to tumor cells after pretreatment with ABN202.

[0028] Figure 4a shows the results of a quantitative analysis of cell surface expression of HMGB1 and HSP70 by ABN202 treatment and heat stimulation on tumor cells.

[0029] Figure 4b shows the results of analyzing the cell surface expression of HMGB1 and HSP70 by ABN202 treatment and heat stimulation on tumor cells through flow cytometry.

[0030] Figure 5a shows the results of analyzing GSH levels in tumor cells by ABN202 treatment and heat stimulation through flow cytometry.

[0031] Figure 5b shows the results of a quantitative analysis of GSH levels in tumor cells by ABN202 treatment and heat stimulation.

[0032] Figure 6a shows the mouse experiment schedule to confirm the efficacy of inhibiting cancer recurrence through the combination of PTT and ABN202.

[0033] Figure 6b shows the results of confirming changes in tumor size, tumor weight, and body weight of mice to verify the efficacy of inhibiting cancer recurrence through the combination of PTT and ABN202.

[0034] Figure 7a shows a mouse experiment schedule for evaluating the effects of immunogenic tumor environment creation and immune cell transplantation through the combination of PTT and ABN202.

[0035] Figure 7b shows the results of confirming changes in tumor size and body weight in mice to confirm the immunogenic tumor environment creation and immune cell transplantation effects through the combination of PTT and ABN202.

[0036] Figure 8a shows an experimental schedule for analyzing immune checkpoint marker expression in immune cells through the combination of PTT and ABN202.

[0037] Figure 8b shows the results of analyzing immune checkpoint markers expressed in immune cells (CD4 T cell, CD8 T cell, Treg cell) after isolating distant tumors following combination therapy.

[0038] Figure 8c shows the results of analyzing immune checkpoint markers expressed in immune cells (CD4 T cell, CD8 T cell, Treg cell) after isolating the spleen following combination therapy.

[0039] Figure 8d shows the results of analyzing immune checkpoint markers expressed in immune cells (CD4 T cell, CD8 T cell, Treg cell) after isolating tumors following combination therapy.

[0040] Figure 9a shows an experimental schedule for analyzing immune cell distribution and activation marker expression through the combination of PTT and ABN202.

[0041] Figure 9b shows the results of analyzing the ratio of immune cells in the spleen after the combined use of PTT and ABN202.

[0042] Figure 9c shows the results of analyzing the ratio of immune cells in distant tumors and activation markers (Granzyme B, IFN-γ) expressed in immune cells after the combination of PTT and ABN202.

[0043] Figure 10 shows the results of analyzing the number of cells per tumor weight after the combined use of PTT and ABN202.

[0044] Figure 11 shows the results of analyzing the ratio of dendritic cells within immune cells and activation markers (CD80, CD86) expressed in dendritic cells in distant tumors, spleens, and tumor drainage lymph nodes (TDLN) after the combination of PTT and ABN202.

[0045] Figure 12 shows the results of analyzing the memory formation rate of T cells after the combined use of PTT and ABN202.

[0046] Figure 13a shows an experimental schedule to confirm the efficacy of treating metastatic tumors through the combination of PTT and ABN202.

[0047] Figure 13b shows the results of monitoring distant tumor growth after the combined use of PTT and ABN202.

[0048] Figure 14a shows an experimental schedule to confirm the difference in therapeutic efficacy for metastatic tumors according to changes in the ABN202 administration schedule when PTT and ABN202 are used in combination.

[0049] Figure 14b shows the results of monitoring distant tumor growth after the initial administration of ABN202 when PTT and ABN202 are used in combination.

[0050] Figure 15a shows the results confirming the tumor cell binding ability of ABN202 and the direct anti-tumor activity resulting therefrom.

[0051] Figure 15b shows the results confirming the tumor cell binding ability of ABN202 and the direct anti-tumor activity resulting therefrom.

[0052] Figure 15c shows the results of flow cytometry analysis to confirm the target binding ability of ABN202.

[0053] Figure 16a shows the results of comparing the direct antitumor activity of a single cytokine conjugate and a double cytokine conjugate.

[0054] Figure 16b shows the results of comparing the direct antitumor activity of a single cytokine conjugate and a double cytokine conjugate.

[0055] Figure 17 shows the results confirming the dose-dependent antitumor effect of ABN202.

[0056] Figure 18a shows a mouse experiment schedule to evaluate the functional contribution of immune cells through the combination of PTT and ABN202.

[0057] Figure 18b shows the results of measuring changes in tumor size and body weight in mice to confirm the functional contribution of immune cells following the combination of PTT and ABN202.

[0058] Figure 19a shows a mouse experiment schedule to evaluate the therapeutic efficacy of PTT, ABN202, and immune checkpoint inhibitors in combination for metastatic tumors.

[0059] Figure 19b shows the results of measuring changes in tumor size and body weight in mice to confirm the therapeutic effect of PTT, ABN202, and immune checkpoint inhibitors on metastatic tumors.

[0060] The present invention will be described in detail below.

[0061] The present invention relates to a variant of human IFN-β and an anti-claudin 3 antibody fusion protein containing the same. More specifically, the present invention provides a fusion protein in which an IFN-β variant having a specific amino acid residue of human IFN-β substituted is combined with an anti-claudin 3 antibody or an antigen-binding fragment thereof.

[0062] According to one aspect of the present invention, a fusion protein comprising an IFN-β variant in which the 17th amino acid of human IFN-β, cysteine, is substituted with serine and the 27th amino acid, arginine, is substituted with threonine is provided.

[0063] In the present invention, IFN-β is a globular protein having a size of about 22 kDa and containing five alpha helix structures, and is known to exhibit various immunological activities such as antiviral activity, cell growth inhibition or antiproliferative activity, lymphocyte cytotoxicity enhancement activity, immunomodulatory activity, target cell differentiation induction or inhibition activity, macrophage activation, increased cytokine production, increased cytotoxic T cell activity, and increased natural killer cell activity. Due to these characteristics, IFN-β has been reported to be useful in the treatment of cancer, autoimmune diseases, viral infections, HIV-related diseases, hepatitis C, and rheumatoid arthritis.

[0064] However, IFN-β is a relatively hydrophobic protein prone to aggregation, and there were limitations in the development and utilization of therapeutic agents due to its low biological activity and productivity, as well as its short half-life. Accordingly, in this invention, a variant was constructed by introducing a site-directed mutagenesis into the IFN-β gene, thereby utilizing a variant with improved physical properties and stability compared to wild-type IFN-β.

[0065] An IFN-β variant according to one embodiment of the present invention may include one or more glycosyl groups. The glycosyl groups may be two or more, and in one embodiment, the IFN-β variant may be configured to undergo glycosylation at asparagine (Asn) residues 25 and 80.

[0066] The above IFN-β variant may be represented by the amino acid sequence of SEQ ID NO. 18 and may retain the biological activity inherent to wild-type IFN-β (SEQ ID NO. 17).

[0067] The fusion protein according to the present invention may include an anti-claudin 3 antibody or an antigen-binding fragment thereof. Generally, antibodies have a Y-shaped structure and consist of two heavy chains and two light chains. Each heavy chain and light chain is connected to the others by disulfide bonds and is divided into a variable region that specifically binds to an antigen and a constant region that performs an effector function. The variable region contains a complementarity-determining region (CDR) that forms a specific binding with the antigen, which is an important structural element that determines the binding specificity and affinity of the antibody.

[0068] The antibody or antibody fragment used in the present invention may include various forms that maintain antigen-binding ability, such as, for example, Fab fragment, Fab' fragment, F(ab')₂ fragment, Fv fragment, scFv, scFv-Fc, diabody, or dsFv, but are not limited thereto.

[0069] In one embodiment of the present invention, the antibody specifically binds to the extracellular loop 2 (ECL2) of claudin 3 and can be internalized into the cell after binding.

[0070] Claudin 3 is a membrane protein that constitutes tight junctions and is known to have four transmembrane regions and contain two extracellular loops (ECL1 and ECL2). In normal tissues, claudin 3 is mainly located inside tight junctions, but in tumor cells, claudin 3 can be exposed on the cell surface due to abnormalities in the tight junction structure. Due to these characteristics, claudin 3 is being suggested as a promising tumor target protein in various cancers.

[0071] According to one embodiment of the present invention, the antibody may include a heavy chain variable region and a light chain variable region comprising the following CDR sequence.

[0072] The heavy chain variable region may include a heavy chain complementarity determining region 1 (VH-CDR1) containing the amino acid sequence of SEQ ID NO. 1, a heavy chain complementarity determining region 2 (VH-CDR2) containing the amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VH-CDR3) containing the amino acid sequence of SEQ ID NO. 3, and the light chain variable region may include a light chain complementarity determining region 1 (VL-CDR1) containing the amino acid sequence of SEQ ID NO. 9, a light chain complementarity determining region 2 (VL-CDR2) containing the amino acid sequence of SEQ ID NO. 10, and a light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence of SEQ ID NO. 11.

[0073] The above antibody may include a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 4 and a light chain variable region sequence containing the amino acid sequence of SEQ ID NO. 12.

[0074] As long as the antibody according to the present invention includes the above-mentioned CDR combination, there are no particular restrictions on its type, and it may be selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. Preferably, it may be an IgG antibody, and the IgG may include subtypes such as IgG1, IgG2, IgG3, or IgG4, but is not limited thereto.

[0075] In addition, the above antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0076] In one embodiment of the present invention, the antibody comprises two heavy chains, and the IFN-β variant may be attached only to the CH3 domain of one of the two heavy chains. In addition, in one embodiment of the present invention, a knob-into-hole structure may be applied to the Fc region of the heavy chain of the antibody to induce the formation of a heterodimer. For example, the CH3 domain of the first heavy chain may include a hole variant, and the CH3 domain of the second heavy chain may include a knob variant. In one embodiment, the heavy chain invariant region containing the hole variant may include the amino acid sequence of SEQ ID NO. 5, and the heavy chain invariant region containing the knob variant may include the amino acid sequence of SEQ ID NO. 6. Additionally, the heavy chain containing the hole variant may include the amino acid sequence of SEQ ID NO. 7, and the heavy chain containing the knob variant may include the amino acid sequence of SEQ ID NO. 8.

[0077] The above IFN-β variant can be linked to the CH3 domain of a heavy chain containing the knob variant or hole variant.

[0078] In the fusion protein of the present invention, the IFN-β variant may be directly linked to an antibody or linked via a peptide linker. The peptide linker is a short amino acid sequence for linking two protein domains, which can minimize structural interference between proteins and maintain functional independence.

[0079] The above linker may be a flexible peptide linker comprising glycine (Gly) and serine (Ser), for example, may include a (Gly₄Ser)n sequence, where n is an integer from 1 to 5. In one embodiment, the linker may include the amino acid sequence of SEQ ID NO. 19. Additionally, in one embodiment, the IFN-β variant connected by a peptide link to the heavy chain containing the hole variant may include the amino acid sequence of SEQ ID NO. 20, and the IFN-β variant connected by a peptide link to the heavy chain containing the knob variant may include the amino acid sequence of SEQ ID NO. 25.

[0080] The anti-claudin 3 antibody-IFN-β fusion protein of the present invention, configured as described above, can selectively bind to tumor cells expressing claudin 3 and effectively deliver the immunomodulatory function of IFN-β to the tumor microenvironment.

[0081] In addition, the present invention provides a polynucleotide encoding the fusion protein.

[0082] In the present invention, “polynucleotide” means a polymer in which a plurality of nucleotides are connected by phosphodiester bonds, and may include a polymer of deoxyribonucleotides or ribonucleotides in a single-stranded or double-stranded form.

[0083] The above polynucleotide may be in the form of DNA or RNA, and may include, for example, genomic DNA, complementary DNA (cDNA), messenger RNA (mRNA), or nucleotide sequences derived therefrom, but is not limited thereto.

[0084] In addition, the above polynucleotide may include not only a nucleotide sequence encoding the amino acid sequence of the fusion protein of the present invention, but also a complementary nucleotide sequence.

[0085] In one embodiment, the polynucleotide may include nucleotide sequences encoding the knob heavy chain, hole heavy chain, and light chain of the anti-claudin 3 antibody, respectively.

[0086] Specifically, the knob heavy chain refers to a heavy chain containing a knob mutation in the CH3 domain of the antibody heavy chain, and the hole heavy chain refers to a heavy chain containing a hole mutation in the CH3 domain. The knob mutation and the hole mutation may be mutations that form mutually complementary stereostructures, thereby causing the two heavy chains to selectively form a heterodimer.

[0087] In addition, a human IFN-β variant may be connected to the heavy chain directly or through a peptide linker, and the linker may be a peptide linker containing glycine (Gly) and serine (Ser).

[0088] Meanwhile, the light chain refers to the light chain of the anti-claudin 3 antibody and may include a nucleotide sequence that binds to the heavy chain to form an antigen-binding site that specifically binds to claudin 3.

[0089] In one embodiment, the nucleotide encoding the hole heavy chain may include the base sequence represented by SEQ ID NO. 21, the nucleotide encoding the knob heavy chain may include the base sequence represented by SEQ ID NO. 22, the nucleotide encoding the hole heavy chain to which the IFN-β variant is connected may include the base sequence represented by SEQ ID NO. 24, and the nucleotide encoding the light chain may include the base sequence represented by SEQ ID NO. 23.

[0090] In addition, the present invention provides an expression vector comprising the polynucleotide.

[0091] In the present invention, “expression vector” means a vector designed to express a fusion protein comprising an IFN-β variant and an anti-claudin 3 antibody or an antigen-binding fragment thereof in a host cell, and means a gene construct comprising a gene sequence and an expression regulatory sequence that are operably linked so that a target gene can be expressed.

[0092] The term “operably linked” above refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein are functionally linked, such that the transcription and translation of the target gene can be regulated by the expression control sequence. Furthermore, the term “expression control sequence” refers to a DNA sequence that regulates the expression of an operably linked polynucleotide in a specific host cell.

[0093] In one embodiment, the expression vector may include polynucleotides encoding the knob heavy chain, hole heavy chain, and light chain of the anti-claudin 3 antibody, respectively. In this case, the hole heavy chain may include a sequence encoding a fusion protein in which a human IFN-β variant is linked directly or through a peptide linker.

[0094] The above expression vector may include expression regulatory elements such as a promoter, an operator, a translation start codon, a translation stop codon, a polyadenylation signal, and an enhancer, and may further include a signal sequence or a leader sequence for membrane targeting or protein secretion as needed.

[0095] In addition, the expression vector may include a selectable marker for selecting a host cell containing the expression vector, and in the case of a replicable vector, it may self-replicate within the host cell or be incorporated into the host genomic DNA by including an origin of replication.

[0096] The above expression vector can be manufactured using gene recombination technology known in the art, and can be produced using enzymes commonly used in the art, such as restriction enzymes and DNA ligases.

[0097] The above expression vector may be various types of vectors usable for foreign gene expression, and may include, but are not limited to, plasmid vectors, cosmid vectors, viral vectors, etc.

[0098] In addition, the present invention provides a host cell transformed with the expression vector.

[0099] In the present invention, a “host cell” refers to a genetically modified cell into which an external polynucleotide or expression vector is introduced so as to express a target protein. The host cell can regulate the expression of the introduced gene or perform post-translational modification, folding, and assembly of the expressed protein.

[0100] Since different host cells can exhibit different characteristics during the translation and post-translational modification processes of proteins, an appropriate host cell can be selected by considering the expression, stability, biological activity, and post-translational modification of the target protein. For example, expression in eukaryotic cells can be advantageous for the production of biologically active proteins by enabling post-translational modifications such as proper protein folding and glycosylation.

[0101] The above host cell may be any of the various cells known in the art capable of stably cloning and expressing the expression vector, but is not limited thereto. For example, prokaryotic cells may include Escherichia coli strains such as Escherichia coli JM109, Escherichia coli BL21 (DE3), Escherichia coli DH5α, Escherichia coli RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X1776, Escherichia coli W3110, etc.; Bacillus strains such as Bacillus subtilis, etc.; Salmonella typhimurium, Serratia marcescens, and Pseudomonas strains, etc.

[0102] In addition, eukaryotic cells such as yeast cells, insect cells, or mammalian cells may be used, for example, yeast cells such as Saccharomyces cerevisiae, or CHO (Chinese hamster ovary), WI-38, BHK, COS-7, HEK293, HepG2, 3T3, RIN, and MDCK cell lines may be used. In one embodiment, the host cell may be a mammalian cell suitable for the expression of an antibody or antibody-fusion protein, and preferably may be a CHO cell.

[0103] The introduction of the above-mentioned expression vector may be carried out using various transformation or transfection methods known in the art. In the present invention, “transformation” or “transfection” refers to a process of introducing external DNA into a host cell so that said DNA is replicated or expressed within the host cell.

[0104] The above transformation or transduction methods may include, but are not limited to, the CaCl₂ precipitation method, the Hanahan method, electroporation, calcium phosphate precipitation method, protoplast fusion method, polyethylene glycol (PEG) method, dextran sulfate method, lipofection method, or Agrobacterium-mediated transformation method.

[0105] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein as an active ingredient.

[0106] The above fusion protein comprises a structure in which an IFN-β variant with enhanced physical stability is combined with an anti-claudin 3 antibody or an antigen-binding fragment thereof, and can selectively bind to tumor cells expressing claudin 3 to induce an anti-tumor immune response. In addition, since the above fusion protein may exhibit immunomodulatory activity, anti-tumor activity, and antibody-dependent cellular cytotoxicity (ADCC), a composition containing it as an active ingredient can be usefully utilized as an anticancer pharmaceutical composition that inhibits the proliferation or activity of cancer cells.

[0107] The above pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipients, diluents, stabilizers, preservatives, isotonic agents, buffers, and other pharmaceutically acceptable adjuvants. These components may be appropriately selected according to methods commonly used in the art.

[0108] The above pharmaceutical composition may be prepared in various formulations according to formulation methods known in the art. For example, the composition may be prepared as an oral formulation such as a powder, granule, tablet, capsule, suspension, emulsion, syrup, or aerosol, or as a parenteral formulation such as a topical preparation, suppository, sterile injectable solution, suspension, emulsion, or lyophilized preparation. In one embodiment, the pharmaceutical composition may be prepared as an injectable preparation.

[0109] The dosage of the above pharmaceutical composition may be appropriately determined by a person skilled in the art according to the patient's age, weight, health condition, type and severity of disease, formulation of the drug, route of administration, and duration of administration. For example, the above composition may be administered once a day or in divided doses in the range of about 0.001 mg / kg to about 1,000 mg / kg, but is not limited thereto.

[0110] The above pharmaceutical composition may be administered via various routes such as oral, intravenous, intramuscular, subcutaneous, abdominal, rectal, or intraventricular, and preferably via a parenteral route.

[0111] In one embodiment of the present invention, the cancer may be a cancer expressing claudin 3, and may include, for example, ovarian cancer, colon cancer, colorectal cancer, bladder cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, breast cancer, prostate cancer, pancreatic cancer, uterine cancer, cervical cancer, melanoma, kidney cancer, or metastatic pleural tumor, but is not limited thereto.

[0112] In the present invention, “prevention” refers to any act of suppressing or delaying the onset of a target disease, and “treatment” refers to any act of improving or alleviating a disease or symptoms related to a disease by administering the pharmaceutical composition of the present invention.

[0113] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein as an active ingredient and used in combination with a phospholipid-photothermal nanoparticle complex having an anti-Claudin-3 antibody or an antigen-binding fragment thereof bound to its surface.

[0114] In the present invention, the photothermal nanoparticles refer to nanoparticles capable of generating heat by absorbing light in the near-infrared region, and may include, for example, polydopamine nanoparticles, gold nanoparticles, graphene nanosheets, or melanin nanoparticles, but are not limited thereto. In one embodiment, the photothermal nanoparticles may be polydopamine nanoparticles formed by the self-polymerization of the compound indopamine represented by the following chemical formula 1. The size of the polydopamine nanoparticles may be about 10 to 500 nm, and preferably about 50 to 200 nm.

[0115]

[0116] The photothermal nanoparticles may be provided in the form of a phospholipid-photothermal nanoparticle complex surrounded by a phospholipid membrane. The phospholipid membrane may comprise, but is not limited to, one or more selected from the group consisting of, for example, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphoglycerol (PG), phosphocholine (PC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-maleimide). Preferably, the DPPC is represented by the following chemical formula 2, the DPPG is represented by the following chemical formula 3, the PG is represented by the following chemical formula 4, the PC is represented by the following chemical formula 5, and the DSPE-PEG2000-maleimide can be represented by the following chemical formula 6.

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] In one embodiment, the phospholipid membrane may comprise DPPC and DPPG, and their molar ratio may be, for example, about 5 to 9:1 to 5, and preferably about 6 to 8:2 to 4. More preferably, they may be mixed in a molar ratio of 6.5 to 7.5:2.5 to 3.5.

[0123] In addition, the phospholipid membrane may include DPPC, DPPG, and DSPE-PEG2000-maleimide, and their molar ratio may be, for example, 5 to 9 : 1 to 5 : 0.01 to 1, and preferably 6 to 8 : 2 to 4 : 0.05 to 0.5. More preferably, they may be included in a molar ratio of 6.5 to 7.5 : 2.5 to 3.5 : 0.05 to 0.2.

[0124] The anti-claudin 3 antibody or its antigen-binding fragment may be bound to the terminal of a pegylated phospholipid membrane. In one embodiment, the antibody or its antigen-binding fragment may be modified to have a free thiol group, and the phospholipid membrane may comprise a phospholipid containing a maleimide group. Accordingly, the antibody or its antigen-binding fragment may be covalently bound to the surface of the phospholipid membrane through a thiol-maleimide reaction.

[0125] In one embodiment, the phospholipid containing the maleimide group may be DSPE-PEG2000-maleimide, but is not limited thereto.

[0126] In one embodiment of the present invention, the anti-claudin 3 antibody or its antigen-binding fragment may be a Claudin 3 antibody or its antigen-binding fragment modified to have free thiol groups. The modified antibody or its antigen-binding fragment means an antibody or its fragment capable of specifically binding to Claudin 3 that has been modified to include free thiol groups, and the modification may include natural modification or artificial modification. For example, the modified Claudin 3 antibody or its binding fragment may include the amino acid sequence of the light chain constant region indicated by SEQ ID NO. 14. More specifically, the Claudin 3 antibody modified to have free thiol groups may include a mutation in which the glutamine (Gln) residue at position 17 of the light chain constant region containing the amino acid sequence of SEQ ID NO. 13 is substituted with cysteine ​​(Cys). The cysteine ​​residue introduced by the above mutation can provide a free thiol group, enabling covalent bonding with phospholipids containing a maleimide group.

[0127] In the present invention, when the photothermal nanoparticles are polydopamine nanoparticles, the phospholipid and polydopamine nanoparticles (PN) may be included in a weight ratio (w / w) of 1 to 20:27, preferably in a weight ratio (w / w) of 5 to 15:27, and more preferably in a weight ratio (w / w) of 7 to 12:27. Most preferably, the phospholipid:polydopamine may be included in a weight ratio (w / w) of 10:27.

[0128] In addition, in the present invention, when the photothermal nanoparticle is a polydopamine nanoparticle, the weight ratio (w / w) of the antibody or a fragment thereof and the polydopamine nanoparticle may be 0.025 to 1:1, preferably 0.1 to 1:1, more preferably 0.3 to 0.7:1, and even more preferably 0.4 to 0.6:1 (w / w). Most preferably, the weight ratio (w / w) of the antibody or a fragment thereof and the polydopamine nanoparticle may be 0.5:1 (w / w).

[0129] The phospholipid-photothermal nanoparticles having the antibody or a fragment thereof bound to their surface may have a particle size of 50 to 300 nm, preferably 100 to 250 nm, and more preferably 120 to 200 nm.

[0130] The phospholipid-photothermal nanoparticle complex with the above-mentioned anti-claudin 3 antibody or its antigen-binding fragment bound to its surface can induce apoptosis of cancer cells upon therapeutically effective light irradiation.

[0131] In the pharmaceutical composition of the present invention, the content of the phospholipid-photothermal nanoparticle complex with the antibody bound to its surface may be about 0.0001 to 99.9 weight% based on the total weight of the composition, preferably about 0.001 to 50 weight%, but is not limited thereto.

[0132] In the pharmaceutical composition of the present invention, the fusion protein and the phospholipid-photothermal nanoparticle complex may be administered simultaneously, separately, or sequentially.

[0133] In addition, the pharmaceutical composition of the present invention may be administered in combination with an immune checkpoint inhibitor.

[0134] The above immune checkpoint inhibitor is a drug that blocks proteins called immune checkpoints expressed in some immune cells, such as T cells, and cancer cells, and may include, but is not limited to, inhibitors or antagonists targeting various immune checkpoints, such as, for example, anti-PD-1 (programmed cell death protein 1) antibody, anti-PD-L1 (programmed cell death ligand 1) antibody, anti-CTLA-4 (cytotoxic T lymphocyte Antigen-4) antibody and anti-TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domain) antibody, anti-LAG-3 (lymphocyte-activation gene 3) antibody, anti-VISTA (V-domain Ig suppressor of T cell activation) antibody, anti-TIM-3 (T cell immunoglobulin and mucin domain-containing protein 3) antibody, or anti-PSGL-1 (P-selectin glycoprotein ligand-1) antibody.

[0135] In addition, the present invention provides a method for preventing or treating cancer, comprising the step of administering an effective amount of the fusion protein to a subject.

[0136] In this invention, “object” refers to mammals including humans.

[0137] In the present invention, “effective amount” means an amount sufficient to produce a beneficial therapeutic effect for the prevention or treatment of cancer in a subject. The effect may include a reduction in the risk of cancer development or a delay in onset, and may include one or more of the following: inhibition of tumor growth, reduction of tumor size, inhibition of metastasis, alleviation of symptoms, or increase in survival rate. The effective amount may vary depending on the condition of the subject and the type of cancer, and may, for example, be in the range of about 0.001 mg / kg to about 1,000 mg / kg.

[0138] In addition, the present invention provides a method for preventing or treating cancer comprising the step of co-administering to a subject the fusion protein and a phospholipid-photothermal nanoparticle complex having an anti-Claudin-3 antibody or an antigen-binding fragment thereof bound to its surface.

[0139] The present invention will be explained in detail below through the following examples and experimental examples.

[0140] However, the following examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited by the following examples and experimental examples.

[0141] <Example 1> Production of recombinant fusion protein

[0142] A recombinant immunocytokine was prepared by fusing an IFN-β variant (mutein) to an anti-claudin 3 (αCLDN3) antibody. The immunocytokine was prepared in two forms: αCLDN3-IFN-β (ABN202), in which an IFN-β variant is fused to one heavy chain, and αCLDN3-2IFN-β, in which an IFN-β variant is fused to each of the two heavy chains (Fig. 1a).

[0143] <1-1> Preparation of αCLDN3-IFN-β (ABN2O2)

[0144] αCLDN3-IFN-β (ABN202) was prepared in which an IFN-β variant was fused to one heavy chain of αCLDN3. The sequence of the recombinant fusion protein is shown in Table 1 below.

[0145]

[0146] Specifically, an IFNβ-C17S / R27T variant was used in which the 17th amino acid residue of IFN-β was substituted with serine (C17S) and the 27th amino acid residue with threonine (R27T). The above IFN-β variant was designed to be fused to the C-terminus of the heavy chain of αCLDN3 via a peptide linker. In addition, a Knob-Hole structure was applied to the Fc region of the heavy chain of the antibody to induce heterodimer formation. That is, structural stability and uniformity were ensured by introducing a “Knob” mutation to one heavy chain and a complementary “Hole” mutation to the other heavy chain to induce the formation of a proper heavy chain dimer. For gene cloning, restriction enzyme cleavage sites of XbaI (TCTAGA) and PacI (TTAATTAA) were inserted into the 5' and 3' ends of the heavy chain gene, respectively. In addition, EcoRI (GAATTC) and AscI (GGCGCGCC) cleavage sites were inserted into the 5' and 3' ends of the light chain gene, respectively. The heavy chain and light chain genes were inserted into the pD2535nt-HDP expression vector (ATUM, Newark, CA, USA) to construct the final expression vector.

[0147] The constructed αCLDN3-IFN-β expression vector was transfected into CHO-K1 cells (Horizon Discovery, Cambridge, UK) using FreeStyle™ MAX reagent (Thermo Scientific). OptiPRO™ SFM medium was added to and mixed with the FreeStyle™ MAX reagent-DNA complex, and the mixture was added to flasks containing CHO-K1 cells and cultured under 5% CO₂ and humidified conditions. 48 hours after transfection, cell lines stably expressing the fusion protein were selected. High-expression cell lines were established through a selection process using 50 μM methionine sulfoximine (MSX). The selected cell lines were cultured for 14 days at 37°C, 5% CO₂, and 125 rpm while supplying glucose to induce fusion protein expression.

[0148] After recovering the fusion protein from the CHO-K1 cell culture supernatant, it was purified using Protein A affinity chromatography. Specifically, the culture supernatant was passed through a column packed with Protein A MabSelect SuRe (Cytiva), impurities were removed using an equilibration buffer and a wash buffer, and αCLDN3-IFN-β (ABN202) was purified using an elution buffer.

[0149] In this way, by attaching an IFNβ-C17S / R27T variant to an antibody platform with a Knob-Hole structure, an immunocytokine ABN202 with optimized structural stability and biological activity was prepared (Fig. 1a).

[0150] <1-2> Preparation of αCLDN3-2IFN-β

[0151] αCLDN3-2IFN-β was produced in which an IFN-β variant was fused to each of the two heavy chains of the anti-claudin 3 (αCLDN3) antibody.

[0152] The above αCLDN3-2IFN-β used the same IFNβ-C17S / R27T variant as in Example 1-1, and was designed so that the IFN-β variant was fused to the C-terminus of the CH3 domain of each of the two heavy chains of the αCLDN3 antibody through a peptide linker.

[0153] The gene cloning, expression vector construction, CHO-K1 cell transduction, cell line selection, and protein purification processes were performed in the same manner as in Example 1-1.

[0154] <Example 2> Preparation of a phospholipid-photothermal nanoparticle complex aCLDN3-Ab-PDA (PTT) with anti-Claudin-3 antibody bound to its surface

[0155] Using an anti-claudin 3 (αCLDN3) antibody variant (h4G3cys) in which a glutamine residue (Q125) is substituted with a cysteine ​​residue, a polydopamine nanoparticle-based photothermal therapeutic complex (aCLDN3-Ab-PDA, PTT) was prepared in which the antibody is covalently bonded to a surface lipid terminal and surrounded by a thin film of a phospholipid layer (Fig. 1b).

[0156] <2-1> Preparation of Anti-Claudin-3 Antibody (h4G3cys)

[0157] h4G3 was selected as an anti-Claudin-3 (αCLDN3) antibody targeting Claudin 3. A single cysteine ​​variant (h4G3cys) was prepared by substituting the glutamine residue at position 125 (Q125) of the light chain of h4G3 (glutamine residue at position 17 of the light chain constant region) with cysteine ​​(SEQ No. 16) to enable site-specific binding to the surface of nanoparticles.

[0158] To establish a cell line that stably expresses h4G3cys, the light chain gene containing the cysteine ​​substitution mutation and the heavy chain gene of h4G3 were each cloned into the Freedom pCHO 1.0 vector (Thermo Fisher Scientific, Inc.). Subsequently, the recombinant expression vector was transfected into Freedom CHO-S cells (Thermo Fisher Scientific, Inc.).

[0159] Transfected CHO-S cells were cultured for 2 weeks under orbital shaker conditions of 130 rpm in a humidified environment of 37°C and 8% CO₂. During the culture period, antibody expression was induced by supplying an additional 4 g / L of glucose on day 3 and day 5, and 6 g / L of glucose on day 7.

[0160] After the culture was finished, the culture supernatant was collected and loaded onto MabSelect SuRe Protein A resin (GE Healthcare, Piscataway) to purify the antibodies by affinity chromatography. The bound antibodies were buffer-eluted and immediately neutralized with 1 mol / L Tris-HCl (pH 8.0) solution. Subsequently, buffer exchange and concentration were performed using an Amicon Ultra-15 centrifugal concentrator (Merck Millipore).

[0161] In this way, a position-specific binding αCLDN3 antibody variant (h4G3cys) containing a single cysteine ​​residue was prepared.

[0162] <2-2> Preparation of Polydopamine Nanoparticles (PDN)

[0163] Polydopamine nanoparticles (PDN) were synthesized through the self-polymerization of dopamine under alkaline conditions.

[0164] Specifically, 50 mg of dopamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 25 mL of triple distilled water (TDW). Subsequently, 1 N sodium hydroxide solution was slowly added dropwise to adjust the pH of the reaction solution to 10, and then magnetically stirred at 50°C for 12 hours. After the reaction was complete, the reaction mixture was centrifuged at 13,500×g for 20 minutes to recover the formed black pellet. Washing with TDW was repeated until the supernatant became clear. After the final wash, the obtained polydopamine nanoparticles (PDN) were resuspended in TDW and stored at 4°C.

[0165] <2-3> Preparation of aCLDN3-Ab-PDA (PTT)

[0166] Hybrid lipid-photothermal nanoparticles (HLPN) were prepared by hydrating a phospholipid thin film using the polydopamine nanoparticles (PDN) prepared above, and then an h4G3cys antibody was attached to the surface to finally prepare aCLDN3-Ab-PDA (PTT).

[0167] Specifically, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-maleimide, ammonium salt) were dissolved in chloroform:methanol (4:1, v / v) at a molar ratio of 7:3:0.1. This was concentrated under reduced pressure to form a uniform lipid thin film.

[0168] Lipid-photothermal nanoparticles (HLPN) were prepared by adding a polydopamine nanoparticle (PDN) suspension prepared in <2-1> above to the formed lipid thin film and hydrating it. Subsequently, an αCLDN3 antibody variant (h4G3cys) containing a single cysteine ​​residue was added to induce a thiol-maleimide reaction between maleimide-PEG lipids and cysteine. The reaction was carried out overnight at 4°C with vigorous stirring.

[0169] After the reaction was complete, lipid-photothermal nanoparticles (Ab-HLPN) with antibodies bound to their surfaces were recovered by centrifugation at 13,500×g for 20 minutes. The recovered particles were resuspended in 1 mL of distilled water and passed through a 400 nm polycarbonate membrane to homogenize the particle size. Finally, the lipid-photothermal nanoparticle complex (aCLDN3-Ab-PDA, PTT) with antibodies covalently bound to its surface was stored at 4°C.

[0170] <Experimental Example 1> Verification of in vitro photothermal therapy efficacy of antibody-photonanoparticle conjugates using mouse tumor cells

[0171] Experiments were conducted to verify the efficacy of photothermal therapy (PTT) induced by antibody-photonanoparticle conjugates in mouse tumor cells.

[0172] Specifically, 1 × 10⁶ mouse colorectal cancer cells expressing mouse CLDN3 per well in a 24-well plate 5After seeding, the cells were cultured for 24 hours at 37°C under 5% CO₂ conditions. Subsequently, control-antibody-PDA and aCLDN3-antibody-PDA (PTT) were added at a concentration of 300 μg / mL each, and the cells were incubated at 37°C for 1 hour. After 1 hour, the cells were washed with DPBS and detached from the plate using cell dissociation buffer. The cells were collected in a 1.5 mL tube and centrifuged at 3,000 rpm for 5 minutes to form a pellet. After removing a portion of the supernatant, the pellet was irradiated with an 808 nm near-infrared (NIR) laser at a power of 1.5 W for 5 minutes. Temperature changes were measured using a thermal imaging camera during irradiation (Fig. 2a).

[0173] Subsequently, cells were resuspended in 1 mL of complete culture media, dispensed at a dose of 100 μL into 96-well plates, and re-cultured for 24 hours. After removing the supernatant, EZ-Cytox (Dogen, #EZ-500) was diluted 10-fold in RPMI medium and administered at a dose of 100 μL. Cell viability was analyzed by measuring absorbance after color development was complete (Fig. 2b).

[0174] As a result, as shown in Figures 2a and 2b, it was confirmed that the temperature in the aCLDN3-antibody-PDA (PTT) treatment group increased significantly compared to the control-antibody-PDA upon NIR irradiation, and cell viability decreased significantly. This implies that CLDN3-targeted photothermal therapy selectively induces tumor cell death.

[0175] <Experimental Example 2> Confirmation of the synergistic effect of ABN202 treatment and heat stimulation on apoptosis

[0176] Experiments were conducted to confirm the synergistic effect of ABN202(αCLDN3-IFN-β) pretreatment and heat stress on apoptosis.

[0177] Specifically, HT-29 cells (human colorectal cancer cells) are placed in a 96-well plate at a rate of 1 x 10⁶ per well. 4 Cells were seeded and cultured for 24 hours. Subsequently, ABN202 was treated with serial dilutions starting from 10 μM in tenfold increments. After 24 hours, heat stress was applied using a water bath at 45°C for 20 minutes. After an additional 48 hours of culture, cell viability was measured by performing the EZ-Cytox assay. The combination effect was analyzed using the combination index (CI) via the Chou-Talalay method (Fig. 3).

[0178] As a result, as shown in Figure 3, it was confirmed that cell viability in the combined treatment group was significantly reduced compared to the single treatment group. In addition, it was confirmed that a synergistic effect exists, with CI values ​​of 1 or higher in a specific concentration range. This suggests that IFN-β signaling amplifies apoptosis induced by heat stimulation.

[0179] <Experimental Example 3> Induction of DAMP expression by ABN202 treatment and thermal stimulation

[0180] Experiments were conducted to determine whether the combined treatment of ABN202 and heat stress induces the expression of damage-associated molecular patterns (DAMPs) associated with immunogenic apoptosis.

[0181] Specifically, 5 x 10 HT-29 cells in a 6-well plate 5 Cells were seeded and treated with 5 nM ABN202 after 24 hours. After 2 hours, the cells were washed and the media was replaced with fresh media. 24 hours after ABN202 treatment, heat stress was applied at 45°C for 20 minutes. After 24 hours, cells were collected, and the expression of HMGB1 and HSP70 was analyzed by flow cytometry.

[0182] As a result, as shown in Figures 4a and 4b, it was confirmed that HMGB1 and HSP70 cell surface expression increased most significantly in the ABN202+Heat combination group. This means that the combination treatment increases the release of DAMPs, thereby promoting immunogenic cell death (ICD).

[0183] <Experimental Example 4> Confirmation of Inhibition of Cell Recovery Ability by ABN202 and Thermal Stimulation

[0184] An experiment was conducted to determine whether thermal stimulation inhibited cell recovery ability after ABN202 pretreatment.

[0185] Specifically, the cell treatment conditions were performed in the same manner as in Experimental Example 3. After culturing for 24 hours following heat stimulation, the cells were collected and the intracellular GSH (Glutathione) levels were analyzed by flow cytometry.

[0186] As a result, as shown in Figures 5a and 5b, it was confirmed that intracellular GSH levels were significantly reduced in the ABN202+Heat combined group. This means that the antioxidant defense mechanism was inhibited, thereby impairing cell recovery ability.

[0187] <Experimental Example 5> Confirmation of Cancer Recurrence Inhibitory Effect by Combination of PTT and ABN202

[0188] An experiment was conducted to determine whether the combination therapy of PTT and ABN202 suppresses distant tumor recurrence.

[0189] Specifically, the MC38_mCLDN3 cell line, a mouse colorectal cancer expressing mouse CLDN3, was injected subcutaneously into the left flank of mice at a rate of 5 x 10⁻³ 5 A primary tumor was formed by injecting 100 µl of cells. The tumor was 100–150 mm 3When [the target] was reached, 0.5 mg / 200 µl of aCLDN3-antibody-PDA (PTT) was administered via the tail vein. At 24, 48, and 72 hours, an 808 nm near-infrared (NIR) laser (1.5 W, 5 min) was irradiated. Subsequently, ABN202 was administered intraperitoneally at a dose of 10 mpk. After 7 days, 5 x 10⁶ identical cells were placed subcutaneously in the right flank. 5 Distal tumors were formed by injecting cells / 100 µl, and tumor growth was monitored for 20 days. At the end of the study, tumor tissue (distal tumor) was isolated from the mice, and its size and weight were measured (Fig. 6a).

[0190] As a result, as shown in Figures 6b to 6d, no change in body weight was observed in any of the experimental groups, and significant inhibition of distal tumor growth and reduction in tumor weight were observed in the combined group. This suggests that local PTT induces a systemic immune response, and ABN202 amplifies this to induce an abscopal effect on the distant tumor.

[0191] <Experimental Example 6> Confirmation of Immunometabolic Induction and Cell Transplantation Effects by Combined Use of PTT and ABN202

[0192] An experiment was conducted to determine whether the combination therapy of PTT and ABN202 induces immune memory.

[0193] Specifically, the tumor formation and PTT / ABN202 treatment methods were performed in the same manner as in Experimental Example 5. On the 7th day after treatment, the spleen was isolated and 1×10 splenocytes were collected. 7 Cells were intravenously injected into tumor-implanted mice. Subsequently, the tumor volume and body weight of the splenocyte-implanted mice were measured to evaluate the therapeutic effect of each group (Fig. 7a).

[0194] As a result, as shown in Figures 7b and 7c, no change in body weight was observed in any of the experimental groups, and it was confirmed that tumor growth inhibition was greatest in the splenocyte transplantation group derived from the combination group. This means that the combination therapy induced tumor-specific adaptive immunity and the formation of immune memory.

[0195] <Experimental Example 7> Confirmation of changes in immune checkpoint marker expression after combination therapy

[0196] Experiments were conducted to confirm changes in immune checkpoint marker expression in immune cells after PTT and ABN202 combination therapy.

[0197] Specifically, the tumor model construction and treatment conditions were performed in the same manner as in Experimental Example 5. One week after the end of treatment, the distal tumor and spleen were isolated, and the expression of PD-1, PD-L1, CTLA-4, and TIGIT in CD4, CD8, and Treg cells was analyzed by flow cytometry (Fig. 8a).

[0198] As a result, as shown in Figures 8b and 8c, an increase in the expression of some immune checkpoint markers was confirmed along with an increase in effector T cell activity in the combined group. This implies that a compensatory regulatory mechanism is at work following strong immune activation.

[0199] <Experimental Example 8> Confirmation of immune cell distribution and activation marker expression after combination therapy

[0200] Experiments were conducted to determine the effects of PTT and ABN202 combination therapy on immune cell distribution and activation marker expression within the tumor microenvironment.

[0201] Specifically, using Human IFNAR1 / 2 knock-in mice, MC38_mCLDN3 cells were injected subcutaneously into each flank at a rate of 5×10⁶ 5Primary and distal tumors were formed by injecting cells / 100 μL. When the tumor reached 100–150 mm³, 0.5 mg / 200 μL of aCLDN3-antibody-PDA was administered via the tail vein (Day 0). One day later, the left primary tumor was irradiated with an 808 nm near-infrared (NIR) laser at 1.5 W for 5 minutes. Subsequently, ABN202 (5 mpk) was administered intraperitoneally on Days 2 and 4, respectively. The control group was administered only aCLDN3-antibody-PDA and did not receive NIR irradiation or ABN202 treatment. One week after the end of treatment, the mice were sacrificed, and the distal tumor and spleen were isolated. After preparing a single-cell suspension, the ratios of CD4 T cells, CD8 T cells, and Treg cells were analyzed via flow cytometry, and Granzyme B and IFN-γ expression in CD8 T cells were measured (Fig. 9a).

[0202] As a result, as shown in Figures 9b and 9c, it was confirmed that the proportion of CD8 T cells in the distal tumor significantly increased in the combination therapy group. In addition, it was confirmed that the expression of Granzyme B and IFN-γ in CD8 T cells significantly increased. This means that the combination therapy promotes the activation of cytotoxic T cells, thereby enhancing the anti-tumor immune response.

[0203] <Experimental Example 9> Analysis of Intratumoral Immune Cell Infiltration After Combination Therapy of PTT and ABN202

[0204] An experiment was conducted to quantitatively analyze the effect of PTT and ABN202 combination therapy on the degree of immune cell infiltration within tumors.

[0205] Specifically, the tumor model construction and treatment conditions were performed in the same manner as in Experimental Example 8. One week after the end of treatment, the distal tumor was isolated, its weight was measured, and single cells were isolated. The absolute number of CD4 T cells, CD8 T cells, and Treg cells was calculated using flow cytometry, and the number of immune cells per tumor weight (cells / mg) was calculated by dividing this by the tumor weight (g).

[0206] As a result, as shown in Figure 10, it was confirmed that the tumor weight decreased in the combination therapy group and the number of CD8 T cells per unit tumor weight significantly increased. This means that the combination therapy promotes immune cell infiltration into the tumor and forms an immune-activated tumor microenvironment.

[0207] <Experimental Example 10> Analysis of Dendritic Cell Activation After Combination Therapy of PTT and ABN202

[0208] Experiments were conducted to determine the effects of PTT and ABN202 combination therapy on dendritic cell activation.

[0209] Specifically, the tumor model construction and treatment conditions were performed in the same manner as in Experimental Example 8. One week after the end of treatment, the distal tumor and tumor drainage lymph nodes (TDLN) were isolated to prepare a single-cell suspension. The proportion of dendritic cells was analyzed by flow cytometry, and the expression of activation markers CD80 and CD86 in the corresponding cells was measured. In addition, the activation level of monocyte-derived dendritic cells (moDC) within the tumor and PD-L1 expression were further analyzed (Fig. 11).

[0210] As a result, as shown in Figure 11, the proportion of DCs within the tumor (DC in tumor, %) increased in the combination therapy group, and in particular, moDC activation increased significantly. The expression of activation markers CD80 and CD86 also increased, confirming that the maturation and functional activation of DCs were promoted. Meanwhile, changes in PD-L1 expression levels in moDCs within the tumor were also observed, suggesting that the combination therapy may also influence immunomodulatory signals within the tumor microenvironment. These results indicate that the combination therapy of PTT and ABN202 can amplify the anti-tumor immune response by inducing the activation of antigen-presenting cells in the tumor microenvironment and enhancing T cell priming.

[0211] <Experimental Example 11> Analysis of Memory T Cell Formation After PTT and ABN202 Combination Therapy

[0212] An experiment was conducted to determine whether the combination therapy of PTT and ABN202 induces Memory T cell formation.

[0213] Specifically, the tumor model construction and treatment conditions were performed in the same manner as in Experimental Example 8. One week after the end of treatment, the spleen was isolated to isolate immune cells. CD44 and CD62L expression in CD4 and CD8 T cells was analyzed by flow cytometry, and based on this, they were classified into Naive (CD44- CD62L+), Effector memory T cell (Tem, CD44+ CD62L-), and Central memory T cell (Tcm, CD44+ CD62L+) (Fig. 12).

[0214] As a result, as shown in Figure 12, it was confirmed that the CD8 Tem and Tcm ratios significantly increased in the combination therapy group. This indicates that combination therapy induces the formation of long-term immune memory and is a treatment strategy with the potential to suppress recurrence.

[0215] <Experimental Example 12> Confirmation of therapeutic efficacy for metastatic tumors through combination therapy of PTT, ABN202, and anti-PD-1 1

[0216] Experiments were conducted to determine whether the triple combination of PTT, ABN202, and anti-PD-1 antibodies inhibits metastatic tumor growth.

[0217] Specifically, 5×10 MC38_mCLDN3 cells in the left subcutaneous tissue of Human IFNAR1 / 2 knock-in mice 5 A primary tumor was formed by injecting 100 μL of cells. One week later, a distal tumor was formed by injecting the same cells subcutaneously into the contralateral side. When the primary tumor reached 100–150 mm³, 0.5 mg / 200 μL of PTT was administered intravenously (Day 1). After 24 and 48 hours, an 808 nm near-infrared (NIR) laser was irradiated at 1.5 W for 5 minutes (Days 2 and 3). ABN202 was administered intraperitoneally at 10 mpk once a week for 2 weeks starting from Day 4. Anti-PD-1 antibody was administered intraperitoneally at 10 mpk three times a week for 2 weeks starting from Day 4. The growth of the distal tumor was monitored until Day 23 (Fig. 13a).

[0218] As a result, as shown in Figures 13b and 13c, no change in body weight was observed in any of the experimental groups, and it was confirmed that the greatest inhibition of distal tumor growth occurred in the triple combination group. This means that the anti-tumor immunity induced by the combination therapy is further amplified through immune checkpoint blockade.

[0219] <Experimental Example 13> Confirmation of therapeutic efficacy for metastatic tumors through combination therapy of PTT, ABN202, and anti-PD-1 2

[0220] An experiment was conducted to confirm the difference in therapeutic efficacy for metastatic tumors according to changes in the ABN202 administration schedule.

[0221] Tumor model construction and PTT administration were performed in the same manner as in Experimental Example 12. ABN202 was administered intraperitoneally at 10 mpk at 24, 48, and 72 hours (Days 2, 3, and 4) consecutively. Anti-PD-1 antibody was administered intraperitoneally at 10 mpk three times a week for 2 weeks starting from Day 4. The growth of the distal tumor was monitored until Day 17 (Fig. 14a).

[0222] As a result, as shown in Figures 14b and 14c, no change in body weight was observed in any of the experimental groups, and it was confirmed that distal tumor growth inhibition occurred early in the initial intensive administration group. This suggests that the intensity of the initial immune activation of ABN202 has a significant influence on the formation of systemic anti-tumor immunity.

[0223] <Experimental Example 14> Evaluation of Tumor Cell Binding Ability and Direct Anti-Tumor Activity of ABN202 (in vitro)

[0224] Experiments were conducted to confirm the tumor cell binding ability of ABN202 and its direct anti-tumor activity.

[0225] Specifically, NCI-H209 cells were seeded in 100 μL of medium at a density of 40,000 per well, NCI-H69, NCI-H146, NCI-H719, and NCI-H889 cells at 20,000 per well, and NCI-H417 cells at 5,000 per well in 96-well plates, and then cultured at 37°C under 5% CO₂ conditions. The test substance was prepared as a double-concentration solution with a maximum concentration of 200 nM, and various concentration ranges were prepared by serially diluting the complete medium in a 1 / 4 ratio. Subsequently, the test substance solution was added to each well to adjust the final volume to 200 μL, and the cells were cultured for an additional 5 days under the same culture conditions. After the culture was finished, 20 μL of WST reagent was added to each well and reacted for 4 hours under light-blocked conditions at 37°C and 5% CO₂. Then, the absorbance at a wavelength of 450 nm was measured to evaluate the cell viability and proliferation inhibitory effects of the test substance treatment.

[0226] In addition, flow cytometry was performed to confirm the target binding ability of ABN202 in cell lines.

[0227] Specifically, recover cultured cells and 2X10 5 Dog cells were treated with ABN501 antibody (10 μg / mL) at 4°C for 1 hour. After washing three times with PBS / 1% FBS buffer, the cells were treated with anti-human IgG-FITC antibody (Jackson ImmunoResearch Laboratories) diluted 1:100 at 4°C for 1 hour. After washing three times again with PBS / 1% FBS buffer, the cells were analyzed using a flow cytometer (BD FACSCalibur).

[0228] As a result, as shown in Table 2 and Figures 15a to 15c, ABN202 exhibited a high binding ability to tumor cells compared to the isotype control group, and accordingly, the reduction in cell viability and the inhibitory effect on proliferation were significantly increased. These results suggest that ABN202 can selectively bind to tumor cells expressing CLDN3 and induce direct anti-tumor activity.

[0229]

[0230] <Experimental Example 15> Comparison of direct antitumor activity of single cytokine conjugate and double cytokine conjugate (in vitro)

[0231] Experiments were conducted to compare the direct antitumor activity based on structural differences in conjugates bound to IFN-β variants.

[0232] Specifically, 3,000 cells were seeded into each well of a 96-well plate and cultured overnight at 37°C under 5% CO₂ conditions. The test substance was prepared to a maximum concentration of 100 nM and serially diluted in a 1 / 4 ratio using complete medium. Subsequently, the existing medium was removed, and the test substance solution was added to adjust the final volume to 200 μL. The cells were then cultured for an additional 5 days under the same culture conditions. Subsequent evaluation of cell viability was performed using the same method as in Experimental Example 14.

[0233] As a result, as shown in Table 3 and Figures 16a and 16b, the tumor cell proliferation inhibitory effect was superior in the single conjugate with one IFN-β compared to the double conjugate with two IFN-βs. These results suggest that a single IFN-β conjugate can induce more efficient signal transduction or cellular responses at the cellular level. In particular, in the case of IFN-β-based immunocytokines, excessive ligand binding may inhibit receptor signal transduction, implying that the single conjugate structure may exhibit more optimized biological activity.

[0234]

[0235] <Experimental Example 16> Evaluation of dose-dependent antitumor effect of ABN202 (in vivo)

[0236] In vivo experiments were performed to confirm the immune-mediated antitumor effect according to the administered dose of ABN202.

[0237] Specifically, MC38 cell lines in which mouse CLDN3 was artificially overexpressed were recovered, washed with DPBS, and 5 × 10⁶ were placed on the right flank of each experimental mouse. 5 Canine cells were injected subcutaneously. ABN202 administration was initiated when the tumor size reached approximately 150 mm³. ABN202 was administered intraperitoneally (IP) at doses of 1, 3, and 10 mg / kg for 2 weeks, with administration repeated three times a week. Tumor size was measured twice a week using digital calipers, and tumor volume was calculated according to the following formula.

[0238] Tumor volume (mm³) = Diameter (mm) × Area (mm²) × 0.5

[0239] Tumor Growth Inhibition (%TGI) was calculated using the following formula.

[0240]

[0241] As a result, as shown in Figure 17, the tumor growth inhibitory effect in the ABN202 administration group increased in a dose-dependent manner as the dosage increased. In particular, the %TGI for the 1 mg / kg, 3 mg / kg, and 10 mg / kg administration groups was approximately 36%, 81%, and 100%, respectively, indicating that the immune-mediated anti-tumor response is enhanced at higher doses. This suggests that a single dose of ABN202 can increase the indirect anti-tumor effect through immune cell activation as the dosage increases, implying that ABN202 may possess dose-dependent therapeutic characteristics in which the anti-tumor effect increases depending on the dosage or administration schedule.

[0242] <Experimental Example 17> Evaluation of Functional Contribution of Immune Cells upon Combination of PTT and IFN-β-Based Immunotherapy in Human IFNAR1 / 2 Knock-in Mice

[0243] Experiments were conducted to investigate the functional roles of major immune cell populations (CD8+ T cells and CD11c+ dendritic cells) contributing to the antitumor effect during combination therapy with PTT and ABN202 in a human IFNAR1 / 2 knock-in mouse model.

[0244] Specifically, a bilateral tumor model was constructed using a colorectal cancer cell line (MC38_mCLDN3) expressing Mouse CLDN3 (Fig. 18a). 5×10⁶ cells were placed subcutaneously in the left flank of the mouse. 5After forming a primary tumor by injecting 100 μL of cells, a distal tumor was formed 7 days later by injecting the same cells at the same concentration into the opposite right flank. When the size of the primary tumor reached approximately 100–150 mm³, PTT was administered once via the tail vein at a dose of 0.5 mg / 200 μL (Day 1). Subsequently, the primary tumor site was irradiated with an 808 nm NIR laser at a power of 1.5 W for 5 minutes, for a total of three additional administrations at 24-hour intervals (Days 2, 3, and 4). ABN202 was administered intraperitoneally at a dose of 10 mg / kg on Days 2, 3, and 4. To evaluate the functional contribution of immune cells, mouse anti-CD8 antibody and mouse anti-CD11c antibody were each administered intraperitoneally at a dose of 5 mg / kg, three times a week for two weeks starting from Day 1. Afterwards, the tumor growth inhibitory effect was evaluated by measuring the size of the distant tumor until Day 22.

[0245] As a result, as shown in Figure 18b, significant inhibition of distant tumor growth was observed in the PTT and ABN202 combination therapy group compared to the control group, demonstrating that local treatment exhibits an abscopal effect that induces a systemic immune response. However, when CD8+ T cells were depleted, the distant tumor suppression effect was significantly reduced, and it was confirmed that the distant tumor suppression effect was also significantly reduced when CD11c+ cells were depleted. This demonstrates that the combination of aCLDN3-antibody-PDA-based photothermal therapy and ABN202 induces potent systemic anti-tumor immunity, and that antigen presentation by CD11c+ dendritic cells and cytotoxic responses by CD8+ T cells play essential roles in this process.

[0246] <Experimental Example 18> Evaluation of therapeutic efficacy of PTT, ABN202, and immune checkpoint inhibitors for the treatment of metastatic tumors

[0247] Experiments were conducted to evaluate whether the co-administration of immune checkpoint inhibitors (anti-PD-1 and anti-CTLA-4) in addition to the combination of PTT and ABN202 in a human IFNAR1 / 2 knock-in mouse model could enhance the antitumor effect against metastatic tumors, and to verify the effect of immune checkpoint blocking strategies on the enhancement of systemic immune responses.

[0248] Specifically, a bilateral tumor model was constructed using a colorectal cancer cell line (MC38_mCLDN3) expressing Mouse CLDN3 (Fig. 19a). 5×10⁶ cells were placed subcutaneously in the left flank of the mouse. 5 A primary tumor was formed by injecting 100 μL of cells, and a distant tumor was formed 7 days later by injecting tumor cells into the right flank under the same conditions. When the size of the primary tumor reached 100–150 mm³, PTT was administered via the tail vein at a dose of 0.5 mg / 200 μL (Day 1). Subsequently, the primary tumor site was irradiated with an 808 nm NIR laser at a power of 1.5 W for 5 minutes at 24-hour intervals for a total of 3 times (Days 2, 3, and 4). ABN202 was administered intraperitoneally at a dose of 10 mg / kg on Days 2, 3, and 4. As immune checkpoint inhibitors, a mouse anti-PD-1 antibody was administered intraperitoneally at a dose of 10 mg / kg three times a week for 2 weeks starting from Day 4, and a mouse anti-CTLA-4 antibody was administered intraperitoneally at a dose of 5 mg / kg at 3-day intervals for a total of 4 times starting from Day 4. Afterwards, the size of the distant tumor was measured until Day 26 to evaluate the therapeutic efficacy.

[0249] As a result, as shown in Figure 19b, inhibition of distant tumor growth was confirmed in the group treated with the combination of PTT and ABN202, and the inhibitory effect on distant tumor growth was significantly increased when anti-PD-1 and anti-CTLA-4 antibodies were additionally combined. In particular, in the group treated with immune checkpoint inhibitors, responses equivalent to tumor regression or complete remission were observed in some individuals, going beyond the delay of distant tumor growth. This demonstrates that tumor antigens are released through aCLDN3-antibody-PDA-based photothermal therapy, and immune activation by ABN202 is amplified by immune checkpoint inhibitors, thereby inducing a powerful systemic immune response that affects distant tumors through local treatment confined to the primary tumor, which can significantly improve therapeutic efficacy against metastatic tumors.

Claims

1. An IFN-β variant in which the 17th amino acid, cysteine, of human interferon-beta (IFN-β) is substituted with serine and the 27th amino acid, arginine, is substituted with threonine; and Comprising an anti-claudin 3 antibody or an antigen-binding fragment thereof, Fusion protein.

2. In Paragraph 1, The above IFN-β variant is characterized by containing a glycosyl group, Fusion protein.

3. In Paragraph 1, The above IFN-β variant is characterized by comprising the amino acid sequence of SEQ ID NO. 18, Fusion protein.

4. In Paragraph 1, The above antibody comprises two heavy chains, and The above IFN-β variant is characterized by being connected to only the CH3 domain of one of the two heavy chains. Fusion protein.

5. In Paragraph 1, The above antibody comprises a first heavy chain and a second heavy chain, and The CH3 domain of the first heavy chain above includes a hole mutation, and The CH3 domain of the second heavy chain above is characterized by including a knob variant, Fusion protein.

6. In Paragraph 5, Characterized by having an IFN-β variant linked to the CH3 domain of the first or second heavy chain, Fusion protein.

7. In Paragraph 6, The above IFN-β variant is characterized by being directly linked to the CH3 domain or linked by a peptide linker. Fusion protein.

8. In Paragraph 7, The above linker is characterized as being a peptide linker comprising glycine (Gly) and serine (Ser). Fusion protein.

9. In Paragraph 8, The above peptide linker is characterized by comprising the amino acid sequence of SEQ ID NO.

19. Fusion protein.

10. In Paragraph 1, The above anti-claudin 3 antibody is characterized by specifically binding to the extracellular loop (ECL) 2 of claudin 3 and being internalized into the cell. Fusion protein.

11. In Paragraph 10, The above anti-claudin3 antibody is A heavy chain variable region comprising a heavy chain complementarity determining site 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO. 1, a heavy chain complementarity determining site 2 (VH-CDR2) comprising the amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining site 3 (VH-CDR3) comprising the amino acid sequence of SEQ ID NO. 3; and Characterized by comprising a light chain variable region including a light chain complementarity determining site 1 (VL-CDR1) containing the amino acid sequence of SEQ ID NO. 9, a light chain complementarity determining site 2 (VL-CDR2) containing the amino acid sequence of SEQ ID NO. 10, and a light chain complementarity determining site 3 (VL-CDR3) containing the amino acid sequence of SEQ ID NO.

11. Fusion protein.

12. A polynucleotide encoding a fusion protein of any one of claims 1 to 1.

13. An expression vector comprising the polynucleotide of claim 12.

14. Host cells transformed with the expression vector of paragraph 13.

15. A fusion protein of any one of claims 1 to 11 comprising as an active ingredient Pharmaceutical composition for the prevention or treatment of cancer.

16. In Paragraph 15, The above cancer is characterized by being selected from the group consisting of ovarian cancer, colon cancer, bladder cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, breast cancer, prostate cancer, pancreatic cancer, uterine cancer, cervical cancer, melanoma, colorectal cancer, kidney cancer, and metastatic pleural tumor. Pharmaceutical composition for the prevention or treatment of cancer.

17. A fusion protein according to any one of claims 1 to 11 is included as an active ingredient, and A phospholipid-photothermal nanoparticle complex in which an anti-Claudin-3 antibody or its antigen-binding fragment is bound to the surface, used in combination with Pharmaceutical composition for the prevention or treatment of cancer.

18. In Paragraph 17, The above anti-Claudin-3 antibody or its antigen-binding fragment is characterized by having a free thiol group in the constant region of the light chain, wherein the free thiol group binds to the maleimide of the phospholipid membrane. Pharmaceutical composition for the prevention or treatment of cancer.

19. In Paragraph 17, The above anti-Claudin-3 antibody is characterized by comprising the amino acid sequence of SEQ ID NO. 14, Pharmaceutical composition for the prevention or treatment of cancer.

20. In Paragraph 17, The above phospholipid-photothermal nanoparticle complex is a phospholipid membrane in which polydopamine nanoparticles, gold nanoparticles, graphene nanosheets, or melanin nanoparticles are captured, and The above phospholipid membrane is i) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-Dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG); ii) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and phosphorylglycerol (PG); iii) phosphocholine (PC) and 1,2-Dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG); iv) phosphocholine (PC) and phosphorylglycerol (PG); and v) comprising any one selected from the group consisting of phosphocholine (PC), and Characterized by further including 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000](DSPE-PEG2000-maleimide), Pharmaceutical composition for the prevention or treatment of cancer.

21. In Paragraph 17, The above phospholipid-photothermal nanoparticle complex is characterized by having a particle size of 100 nm to 250 nm, Pharmaceutical composition for the prevention or treatment of cancer.

22. In Paragraph 17, The above photothermal nanoparticle composite is characterized by absorbing light in the near-infrared region. Pharmaceutical composition for the prevention or treatment of cancer.

23. In Paragraph 20, Characterized by the above DPPC and DPPG being included in a molar ratio of 5 to 9 : 1 to 5. Pharmaceutical composition for the prevention or treatment of cancer.

24. In Paragraph 20, Characterized by the above DPPC, DPPG, and DSPE-PEG2000-maleimide being included in a molar ratio of 5 to 9 : 1 to 5 : 0.01 to 1. Pharmaceutical composition for the prevention or treatment of cancer.

25. In Paragraph 17, The above phospholipid membrane is characterized by being PEGylated, Pharmaceutical composition for the prevention or treatment of cancer.

26. In Paragraph 17, The above cancer is characterized by being selected from the group consisting of ovarian cancer, colon cancer, bladder cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, breast cancer, prostate cancer, pancreatic cancer, uterine cancer, cervical cancer, melanoma, colorectal cancer, kidney cancer, and metastatic pleural tumor. Pharmaceutical composition for the prevention or treatment of cancer.

27. In Paragraph 17, The above fusion protein and phospholipid-photothermal nanoparticle complex are characterized by being administered simultaneously, separately, or sequentially. Pharmaceutical composition for the prevention or treatment of cancer.

28. In Paragraph 17, The above pharmaceutical composition is characterized by being administered in combination with an immune checkpoint inhibitor. Pharmaceutical composition for the prevention or treatment of cancer.

29. In Paragraph 25, The above immune checkpoint inhibitor, fusion protein, and phospholipid-photothermal nanoparticle complex are characterized by being administered simultaneously, separately, or sequentially. Pharmaceutical composition for the prevention or treatment of cancer.

30. In Paragraph 25, The above immune checkpoint inhibitor is characterized by being selected from the group consisting of anti-PD-1 (programmed cell death protein 1) antibody, anti-PD-L1 (programmed cell death ligand 1) antibody, anti-CTLA-4 (cytotoxic T lymphocyte Antigen-4) antibody and anti-TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domain) antibody, anti-LAG-3 (lymphocyte-activation gene 3) antibody, anti-VISTA (V-domain Ig suppressor of T cell activation) antibody, anti-TIM-3 (T cell immunoglobulin and mucin domain-containing protein 3) antibody, or anti-PSGL-1 (P-selectin glycoprotein ligand-1) antibody. Pharmaceutical composition for the prevention or treatment of cancer.

31. A method for preventing or treating cancer, comprising the step of administering a fusion protein of any one of claims 1 to 11 to a subject.

32. A method for preventing or treating cancer, comprising the step of administering to a subject a fusion protein of any one of claims 1 to 11 and a phospholipid-photothermal nanoparticle complex having an anti-Claudin-3 antibody or an antigen-binding fragment thereof bound to its surface.

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