Fusion protein comprising antibody specifically binding to trop2 and interferon-beta variant, and pharmaceutical composition comprising same

Abstract

The present invention relates to a fusion protein comprising an interferon-beta variant and an antibody that specifically binds to TROP2, and a pharmaceutical composition comprising same. The fusion protein of the present invention can induce direct death of tumor cells and, at the same time, can effectively enhance an anti-tumor immune response, and thus can be effectively used as a therapeutic strategy for preventing or treating cancer.

Description

A fusion protein comprising an antibody that specifically binds to TROP2 and an interferon-beta variant, and a pharmaceutical composition comprising the same.

[0001] The present invention relates to a fusion protein comprising an antibody that specifically binds to TROP2 for the prevention or treatment of cancer and an interferon-beta variant, and a pharmaceutical composition comprising said fusion protein.

[0002] Recently, in the field of targeted tumor therapy, targeted therapy strategies that deliver therapeutic substances by selectively recognizing specific tumor antigens are being actively researched. This approach is attracting attention as a promising therapeutic strategy capable of complementing the limitations of existing chemotherapy-based treatments, as it offers the advantage of increasing selectivity for tumor cells while reducing toxicity to normal tissues. In particular, the development of antibody-based therapies that utilize antibodies to selectively deliver cytotoxic drugs or functional proteins to tumor tissues is actively underway.

[0003] In this regard, TROP2 (Trophoblast cell surface antigen 2) is known as a type I transmembrane glycoprotein that is overexpressed in various epithelial cancers and is encoded by the TACSTD2 gene. TROP2 is involved in the regulation of cell signaling pathways associated with EpCAM-induced signaling and has been reported to play a significant role in the proliferation, invasion, and metastasis of tumor cells. Furthermore, TROP2 overexpression is known to be associated with poor prognosis in several solid tumors, making it a promising therapeutic target for tumor targeted therapy.

[0004] Accordingly, various therapeutic strategies targeting TROP2 are being developed, most notably antibody-drug conjugates (ADCs). ADCs are therapeutic agents that exhibit anticancer effects by selectively delivering drugs to tumor cells through the attachment of cytotoxic drugs to antibodies that recognize tumor antigens. In fact, drugs such as Sacituzumab govitecan and Datopotamab deruxtecan, which are TROP2-targeting ADCs, have been developed and reported to demonstrate clinical efficacy in some types of cancer.

[0005] However, despite these ADC-based therapies demonstrating excellent targeting capabilities for tumor cells, Grade 3 or Grade 4 adverse events have been reported due to toxicity caused by cytotoxic payloads, and the problem of limited duration of response in some patients has been raised. Additionally, limitations have been reported in which therapeutic efficacy may be limited by the development of drug resistance and immunosuppressive mechanisms in the tumor microenvironment.

[0006] Meanwhile, interferon-beta (IFN-β) is known as a cytokine that inhibits tumor cell proliferation and induces apoptosis; at the same time, it has been reported to enhance anti-tumor immune responses by activating antigen-presenting cells and promoting T cell responses. Therefore, immunotherapeutic strategies utilizing IFN-β are presented as a promising approach for inducing anti-tumor immune responses in the tumor microenvironment.

[0007] However, it is known that IFN-β faces difficulties in application as a therapeutic agent due to potential side effects such as immune hyperreactions and systemic toxicity upon systemic administration, as well as the problem of low yield during the protein production process. Accordingly, various variants are being developed to improve the safety and productivity of IFN-β.

[0008] In this regard, an IFN-β variant (C17S / R27T IFN-β mutein) in which the 17th cysteine ​​residue of human IFN-β is substituted with a serine residue and the 27th arginine residue is substituted with a threonine residue has been reported as a variant with improved protein productivity and expression yield. However, therapeutic strategies capable of inducing a potent anti-tumor effect by simultaneously achieving tumor-specific targeting and immune activation have not yet been sufficiently established.

[0009] Accordingly, the inventors designed and manufactured an antibody-cytokine fusion protein by fusing an IFN-β variant to an antibody targeting TROP2, and confirmed that by selectively delivering IFN-β to tumor tissue, it is possible to induce direct apoptosis of tumor cells while simultaneously effectively enhancing the anti-tumor immune response. Furthermore, by optimizing the structure and morphology of the fusion protein, they selected an antibody-cytokine fusion protein possessing optimal efficacy and stability, which were not addressed in the prior art. Through this, the present invention was completed by confirming that it exhibits enhanced anticancer efficacy and improved safety compared to existing TROP2-targeted ADCs.

[0010] The object of the present invention is to provide 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; and an anti-TROP2 antibody or an antigen-binding fragment thereof.

[0011] 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.

[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.

[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] In order to achieve the above objective,

[0015] 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-TROP2 antibody or an antigen-binding fragment thereof.

[0016] 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.

[0017] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein as an active ingredient.

[0018] 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.

[0019] The present invention relates to a fusion protein comprising an antibody that specifically binds to TROP2 and an IFN-β variant, and a pharmaceutical composition comprising the same. The fusion protein of the present invention can induce direct death of tumor cells while simultaneously effectively enhancing an anti-tumor immune response, and thus can be usefully employed as a therapeutic strategy for the prevention or treatment of cancer.

[0020] Figure 1a is a schematic diagram showing the structure of a fusion protein in which IFN-β mutein is bound to a TROP2 target antibody.

[0021] Figure 1b shows the results of comparing the in vitro cancer cell killing ability of αTROP2-IFβm (mono) and αTROP2-IFNβm (dual).

[0022] Figure 2a is a schematic diagram of a human IFNAR1 / 2 knock-in mouse model.

[0023] Figure 2b shows the results of evaluating the in vivo tumor growth inhibitory ability according to the form (mono, dual, or afucosylated) of αTROP2-IFNβm.

[0024] Figure 2c shows the results of confirming changes in mouse body weight according to the form (mono, dual, or afucosylated) of αTROP2-IFNβm.

[0025] Figure 2d shows tumor growth curves according to the morphology (mono, dual, or afucosylated) of αTROP2-IFNβm.

[0026] Figure 3 shows the results of evaluating hematological toxicity indicators according to αTROP2-IFNβm (Mono) and αTROP2-IFNβm (Dual).

[0027] Figure 4 shows the results of comparing the in vivo drug half-lives of αTROP2-IFNβm (Mono) and αTROP2-IFNβm (Dual).

[0028] Figure 5a shows the results of evaluating the cancer cell growth inhibitory ability of ABN202 (αTROP2) in triple-negative breast cancer.

[0029] Figure 5b shows the results of evaluating the cancer cell growth inhibitory ability of ABN202 (αTROP2) in hormone receptor-positive (HR+) breast cancer.

[0030] Figure 5c shows the results of evaluating the cancer cell growth inhibitory ability of ABN202 (αTROP2) in EGFR-mutated non-small cell lung cancer.

[0031] Figure 6a is a schematic diagram showing the construction of an Enhertu (Trastuzumab deruxtecan) resistant cell line.

[0032] Figure 6b shows the results of confirming the mRNA and protein expression levels of ABC transporters (drug efflux pumps) in Enhertu-resistant cell lines.

[0033] Figure 6c shows the results of the recovery of cancer cell killing ability when an ABC transporter inhibitor and an ADC (Enhertu or Dato-Dxd) were treated in combination in Enhertu-resistant cell lines.

[0034] Figure 6d shows the results of comparing the cancer cell killing ability of ADC or ABN202 (αTROP2) in Enhertu-resistant cell lines.

[0035] Figure 6e shows the results of comparing the cancer cell killing ability of Payload, ADC, and ABN202 (αTROP2) in Paclitaxel-resistant cell lines.

[0036] Figure 7a shows the results of evaluating the efficacy of ABN202 (αTROP2) in an MDA-MB-231 triple-negative breast cancer xenograft mouse model.

[0037] Figure 7b shows the long-term observation results of the efficacy of ABN202 (αTROP2) in an MDA-MB-231 triple-negative breast cancer xenograft mouse model.

[0038] Figure 8a shows the results of confirming TROP2 expression levels in an ADC-resistant breast cancer PDX mouse model.

[0039] Figure 8b shows the results of evaluating the efficacy of ABN202 (αTROP2) in an ADC-resistant breast cancer PDX mouse model.

[0040] Figure 9a is a schematic diagram of a comparative experiment on immune cell-mediated anticancer activity.

[0041] Figure 9b shows the results of comparing the cancer cell killing ability of ABN202 (αTROP2) and ADC through co-culture of human immune cells and cancer cells.

[0042] Figure 9c shows the results of comparing the change in the activity level of immune cells according to ABN202 (αTROP2) and ADC through co-culture of human immune cells and cancer cells.

[0043] Figure 10a shows the results of inhibiting hTROP2-MB49 cell growth with ABN202 (αTROP2) or ADC.

[0044] Figure 10b shows the results of inhibiting hTROP2-MB49 cell growth according to the immuno-anticancer efficacy of ABN202 (αTROP2).

[0045] Figure 10c shows the results of evaluating the in vivo immuno-anticancer efficacy of ABN202 (αTROP2) using an hTROP2-MB49 tumor model.

[0046] Figure 11a shows the results of comparing the in vivo immuno-anticancer efficacy of ABN202 (αTROP2) and non-targeted IFNβm using an hTROP2-B16F10 tumor model.

[0047] Figure 11b shows the results of comparing the in vivo immuno-anticancer efficacy of ABN202 (αTROP2) against Control-IFNβm using the hTROP2-B16F10 tumor model.

[0048] Figure 12 shows the results of evaluating the dose-dependent efficacy of ABN202 (αTROP2) using an hTROP2-MB49 tumor model.

[0049] Figure 13 shows the results of comparing the anticancer efficacy of ABN202 (αTROP2) with a combination therapy of ADC and immune checkpoint inhibitor.

[0050] Figure 14a shows the results of confirming the receptor binding ability of IFNβm under various pH conditions.

[0051] Figure 14b shows the results of confirming the cell signaling ability of IFNβm under neutral and acidic pH conditions.

[0052] Figure 14c shows the results of confirming the cell signaling ability of ABN202 (αTROP2) under neutral and acidic pH conditions.

[0053] Figure 15 shows the results of comparing the efficacy of type 1 interferon in a mouse bladder cancer allograft model.

[0054] Figure 16a shows the results of analyzing the immuno-oncology mechanism of ABN202 (αTROP2).

[0055] Figure 16b shows the results of analyzing the activity of immune cells within a tumor by ABN202 (αTROP2).

[0056] Figure 16c shows the results of analyzing immune cell activity in tumor drainage lymph nodes by ABN202 (αTROP2).

[0057] Figure 17a shows the results of analyzing whether long-term anti-tumor memory immunity induced by ABN202 (αTROP2) is formed.

[0058] Figure 17b is a figure showing the results of confirming whether adaptive immune cells were delivered.

[0059] The present invention will be described in detail below.

[0060] The present invention relates to a variant of human IFN-β and an anti-TROP2 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-TROP2 antibody or an antigen-binding fragment thereof.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The above IFN-β variant may be represented by the amino acid sequence of SEQ ID NO. 16 and may retain the biological activity inherent to wild-type IFN-β (SEQ ID NO. 15).

[0066] The fusion protein according to the present invention may include an anti-TROP2 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 other by a disulfide bond 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.

[0067] 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.

[0068] TROP2 is known as Trophoblast cell surface antigen 2, and is also known by other names such as TACSTD2 (Tumor-associated calcium signal transducer 2), EGP-1 (Epithelial Glycoprotein-1), and GA733-1. This protein is a cell surface glycoprotein encoded by the TACSTD2 gene; it is primarily expressed in epithelial cells and is known to be overexpressed in various solid tumors, including breast cancer (particularly triple-negative), lung cancer, gastric cancer, colorectal cancer, and pancreatic cancer. Located on the cell membrane, TROP2 is involved in calcium signaling and plays a crucial role in cell proliferation, migration, invasion, tumorigenesis, and metastasis. Although its expression is limited in normal tissues, it is expressed at high levels in various solid tumors, making it a focal point for cancer diagnosis and treatment.

[0069] In the present invention, the anti-TROP2 antibody may be any antibody known to specifically bind to TROP2, for example, Sacituzumab (hRS7) or datopotamab, but is not limited thereto.

[0070] In one embodiment, the antibody may include a heavy chain variable region and a light chain variable region comprising the following CDR sequence.

[0071] 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.

[0072] The light chain of the above antibody may include the amino acid sequence of SEQ ID NO. 14, and may consist of a light chain variable region of SEQ ID NO. 12 and a light chain constant region of SEQ ID NO. 13. Additionally, the heavy chain of the above antibody may include a heavy chain variable region of SEQ ID NO. 4.

[0073] 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.

[0074] In addition, the above antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0075] 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. Additionally, in one embodiment of the present invention, a knob-into-hole structure may be applied to the Fc region of the antibody's heavy chain to induce the formation of a heterodimer. For example, the CH3 domain of the first heavy chain may contain a hole variant, and the CH3 domain of the second heavy chain may contain a knob variant.

[0076] In one embodiment, the heavy chain constant region of the antibody may include an Fc sequence (G1m1) derived from trastuzumab. In this case, the heavy chain constant region containing the hole mutation may include the amino acid sequence of SEQ ID NO. 5, and the heavy chain constant region containing the knob mutation may include the amino acid sequence of SEQ ID NO. 6. Additionally, the heavy chain containing the hole mutation may include the amino acid sequence of SEQ ID NO. 7, and the heavy chain containing the knob mutation may include the amino acid sequence of SEQ ID NO. 8.

[0077] In another embodiment, the heavy chain constant region of the antibody may include an Fc sequence (G1m3) derived from Sacituzumab. In this case, the heavy chain constant region containing the hole mutation may include the amino acid sequence of SEQ ID NO. 26, and the heavy chain constant region containing the knob mutation may include the amino acid sequence of SEQ ID NO. 27. Additionally, the heavy chain containing the hole mutation may include the amino acid sequence of SEQ ID NO. 28, and the heavy chain containing the knob mutation may include the amino acid sequence of SEQ ID NO. 29.

[0078] The above IFN-β variant can be linked to the CH3 domain of a heavy chain containing the knob variant or hole variant.

[0079] 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.

[0080] The above linker may be a flexible peptide linker comprising glycine (Gly) and serine (Ser), for example, may include a (Gly₄Ser)n sequence, wherein n may be an integer from 1 to 5. In one embodiment, the linker may include the amino acid sequence of SEQ ID NO. 17.

[0081] In one embodiment, when the heavy chain constant region of the antibody includes a G1m1 allotype, an IFN-β variant connected by a peptide link to the heavy chain including the hole variant may include the amino acid sequence of SEQ ID NO. 18, and an IFN-β variant connected by a peptide link to the heavy chain including the knob variant may include the amino acid sequence of SEQ ID NO. 23.

[0082] In another embodiment, when the heavy chain constant region of the antibody includes a G1m3 allotype, an IFN-β variant connected by a peptide link to the heavy chain including the hole variant may include the amino acid sequence of SEQ ID NO. 30, and an IFN-β variant connected by a peptide link to the heavy chain including the knob variant may include the amino acid sequence of SEQ ID NO. 31.

[0083] The anti-TROP2 antibody-IFN-β fusion protein of the present invention, configured as described above, can selectively bind to tumor cells expressing TROP2 and effectively deliver the immunomodulatory function of IFN-β to the tumor microenvironment.

[0084] In addition, the present invention provides a polynucleotide encoding the fusion protein.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] In one embodiment, the polynucleotide may include nucleotide sequences encoding the knob heavy chain, hole heavy chain, and light chain of the anti-TROP2 antibody, respectively.

[0089] 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.

[0090] 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).

[0091] Meanwhile, the light chain refers to the light chain of an anti-TROP2 antibody and may include a nucleotide sequence that binds to the heavy chain to form an antigen-binding site that specifically binds to TROP2. The nucleotide encoding the light chain may include the base sequence indicated by SEQ ID NO. 21.

[0092] In one embodiment, when the heavy chain constant region of the antibody includes a G1m1 allotype, the nucleotide encoding the hole heavy chain may include the base sequence indicated by SEQ ID NO. 19, and the nucleotide encoding the knob heavy chain may include the base sequence indicated by SEQ ID NO. 20. Additionally, the nucleotide encoding the hole heavy chain linked to the IFN-β variant may include the base sequence indicated by SEQ ID NO. 22, and the nucleotide encoding the knob heavy chain linked to the IFN-β variant may include the base sequence indicated by SEQ ID NO. 24.

[0093] In another embodiment, when the heavy chain constant region of the antibody includes a G1m3 allotype, the nucleotide encoding the hole heavy chain may include the base sequence indicated by SEQ ID NO. 32, and the nucleotide encoding the knob heavy chain may include the base sequence indicated by SEQ ID NO. 33. Additionally, the nucleotide encoding the hole heavy chain to which the IFN-β variant is linked may include the base sequence indicated by SEQ ID NO. 34, and the nucleotide encoding the knob heavy chain to which the IFN-β variant is linked may include the base sequence indicated by SEQ ID NO. 35.

[0094] In addition, the present invention provides an expression vector comprising the polynucleotide.

[0095] In the present invention, the term “expression vector” refers to a vector designed to express a fusion protein comprising an IFN-β variant and an anti-TROP2 antibody or an antigen-binding fragment thereof in a host cell, and refers to a gene construct comprising an operably linked gene sequence and an expression regulatory sequence to enable the expression of a target gene.

[0096] 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.

[0097] In one embodiment, the expression vector may include polynucleotides encoding the knob heavy chain, hole heavy chain, and light chain of the anti-TROP2 antibody, respectively. In this case, the knob or 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] In addition, the present invention provides a host cell transformed with the expression vector.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] In addition, eukaryotic cells may include yeast cells, insect cells, or mammalian cells, 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. 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.

[0107] 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.

[0108] 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.

[0109] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein as an active ingredient.

[0110] The above fusion protein comprises a structure in which an IFN-β variant with enhanced physical stability is combined with an anti-TROP2 antibody or an antigen-binding fragment thereof, and can selectively bind to tumor cells expressing TROP2 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] In one embodiment of the present invention, the cancer may be a cancer expressing TROP2, and may include, but is not limited to, breast cancer, triple-negative breast cancer, colorectal cancer, colorectal cancer, head and neck cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, stomach cancer, pancreatic cancer, liver cancer, biliary tract cancer, gallbladder cancer, esophageal cancer, kidney cancer, bladder cancer, ureteral cancer, urothelial carcinoma, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine cancer, testicular cancer, thyroid cancer, parathyroid cancer, skin cancer, melanoma, brain cancer, brain and spinal cord tumor, thymoma, mesothelioma, lymphoma, blood cancer, acute leukemia, chronic leukemia, multiple myeloma, myelodysplastic syndromes (MDS), myelofibrosis, endocrine cancer, and sarcoma.

[0116] 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.

[0117] 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.

[0118] In this invention, “object” refers to mammals including humans.

[0119] 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.

[0120] The present invention will be explained in detail below through the following examples and experimental examples.

[0121] 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.

[0122] <Example 1> Production of recombinant fusion protein

[0123] A recombinant immunocytokine was constructed by fusing an IFN-β variant (mutein) to a TROP2 target antibody. The immunocytokine was produced in two forms: αTROP2-IFNβm (Mono), in which an IFN-β variant is fused to one heavy chain, and αTROP2-IFNβm (Dual), in which an IFN-β variant is fused to each of the two heavy chains (Fig. 1a).

[0124] <1-1> Preparation of αTROP2-IFNβm (Mono)

[0125] αTROP2-IFN-βm (Mono) was prepared in which an IFN-β variant was fused to one heavy chain of αTROP2. The sequence of the recombinant fusion protein is shown in Table 1 below.

[0126]

[0127] 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 IFN-β variant was designed to be fused to the C-terminus of the heavy chain of αTROP2 via a peptide linker. In addition, the antibody applied a Knob-Hole structure to the Fc sequence derived from trastuzumab 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.

[0128] For gene cloning, restriction enzyme XbaI (TCTAGA) and PacI (TTAATTAA) cleavage sites were inserted into the 5' and 3' ends of the heavy chain gene, respectively. Additionally, EcoRI (GAATTC) and AscI (GGCGCGCC) cleavage sites were inserted into the 5' and 3' ends of the light chain gene, respectively. The heavy and light chain genes were inserted into the pD2535nt-HDP expression vector (ATUM, Newark, CA, USA) to construct the final expression vector.

[0129] The constructed αTROP2-IFNβm (Mono) 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.

[0130] 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 αTROP2-IFNβm (Mono) was purified using an elution buffer.

[0131] In this way, by attaching an IFNβ-C17S / R27T variant to an antibody platform with a Knob-Hole structure, an immunocytokine αTROP2-IFNβm (Mono) with optimized structural stability and biological activity was prepared.

[0132] The control group αTROP2-IFNβm (Mono, IgG1 null) was prepared by the same manufacturing method as above after transfecting CHO-K1 cells with an expression vector containing an IgG1 gene modified to remove Fc function.

[0133] <1-2> Preparation of αTROP2-IFNβm (Dual)

[0134] αTROP2-IFNβm (Dual) was constructed in which an IFN-β variant was fused to each of the two heavy chains of the αTROP2 antibody.

[0135] The above αTROP2-IFNβm (Dual) 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 αTROP2 antibody through a peptide linker.

[0136] 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.

[0137] <Experimental Example 1> Comparison of in vitro cancer cell killing ability of αTROP2-IFNβm (Dual) and TαTROP2-IFNβm (Mono)

[0138] Experiments were performed to confirm the in vitro cancer cell growth inhibitory ability of αTROP2-IFNβm (Dual) and αTROP2-IFNβm (Mono).

[0139] Specifically, human breast cancer cell line HCC1954 was seeded at a density of 3,000 cells per well in a 96-well plate (SPL, #30096), and after 16 hours, αTROP2-IFNβm (Dual) and αTROP2-IFNβm (Mono) drugs were administered by serial dilution starting from 100 nM in 1 / 10 increments. After 120 hours had passed since drug treatment, 10 μL of EZ-Cytox (Dogen, #EZ-500) was administered per well, and after 4 hours, the absorbance was measured at a wavelength of 450 nm using a spectrophotometer (Tecan, Spark® Multimode Microplate Reader).

[0140] As a result, as shown in Figure 1b and Table 1, αTROP2-IFNβm (Mono) exhibited a maximum cancer cell growth inhibitory effect similar to that of αTROP2-IFNβm (Dual).

[0141]

[0142] Specifically, the maximum inhibition rates of αTROP2-IFNβm (Dual) and αTROP2-IFNβm (Mono) were confirmed to be 92.6% and 93.6%, respectively, confirming that both drugs exhibit a high level of cancer cell growth inhibitory effect. However, response curve analysis revealed that the IC50 of αTROP2-IFNβm (Mono) 50 The value is 1.592 × 10⁻⁶ -6 It was shown as M, and the IC of αTROP2-IFNβm (Dual) 50 The value is 2.252×10 -5 It showed a value approximately 14 times lower than M. This means that αTROP2-IFNβm (Mono) can induce inhibition of cancer cell growth at lower concentrations while exhibiting the same level of maximum anticancer effect, and suggests that it exhibits higher potency under in vitro conditions compared to αTROP2-IFNβm (Dual).

[0143] <Experimental Example 2> Evaluation of Antitumor Efficacy of αTROP2-IFNβm Fusion Protein in hIFNAR1 / 2 Knock-in Mouse Tumor Model

[0144] Mouse experiments were conducted to compare and evaluate the antitumor efficacy of various αTROP2-IFNβm fusion proteins through changes in the number of IFNβm fusions and antibody Fc function.

[0145] Specifically, since human IFNβ does not have interspecies cross-reactivity with mouse IFNα receptors, human IFNAR1 / 2 knock-in (hIFNAR1 / 2 KI) mice were constructed by substituting the extracellular domains of mouse IFNα receptor 1 and 2 with the extracellular domains of human IFNα receptor 1 and 2, respectively, to evaluate the in vivo activity of human IFNβ, and the antitumor efficacy of the αTROP2-IFNβm fusion protein was evaluated (Fig. 2a).

[0146] To evaluate antitumor efficacy, the MB49 cell line (hTROP2-MB49) overexpressing human TROP2 was administered to hIFNAR1 / 2 KI mice at a rate of 1 × 10⁶ per mouse. 6 Tumors were formed by injecting cells subcutaneously (SC) into the right flank. Tumor size was measured three times a week using a caliper, and tumor volume was calculated using the formula (long axis × short axis²) / 2. When the tumor size reached approximately 100–150 mm³, mice were randomly assigned to the experimental group, and the test substance was administered intraperitoneally (IP) at a dose of 5 mg / kg. The drug was administered once weekly (QW) for a total of three weeks.

[0147] As a result, as shown in Fig. 2b, TROP2-IFNβm (Mono, IgG1 wildtype) exhibited the best tumor growth inhibitory effect among the tested candidate substances. In addition, no significant weight loss was observed during the drug administration period, confirming good tolerability (Fig. 2c). Furthermore, analysis of the tumor response revealed that for TROP2-IFNβm (Mono, IgG1 wildtype), partial response (PR) was observed in 1 out of 6 mice and complete response (CR) in 2 mice (Fig. 2d). These results suggest that TROP2-IFNβm (Mono, IgG1 wildtype) can demonstrate excellent anti-tumor efficacy in in vivo tumor models.

[0148] <Experimental Example 3> Evaluation of Hematological Toxicity of αTROP2-IFNβm Fusion Protein in hIFNAR1 / 2 Knock-in Mice

[0149] Experiments using hIFNAR1 / 2 KI mice were performed to evaluate the hematological toxicity of αTROP2-IFNβm (Dual) and αTROP2-IFNβm (Mono).

[0150] Specifically, αTROP2-IFNβm (Dual) or αTROP2-IFNβm (Mono) was administered as a single intraperitoneal (IP) dose of 5 mg / kg to healthy hIFNAR1 / 2 KI mice. Subsequently, blood was collected from the mice over time, and a complete blood count (CBC) was performed using a blood biochemistry analyzer (Zoetis, Vetscan® VS2). The CBC analysis items included white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), platelets (PLT), and white blood cell fractions including neutrophils (NEU), lymphocytes (LYMPH), and monocytes (MONOCYTE).

[0151] As a result, as shown in Figure 3, mild, reversible hematological changes were observed in both the αTROP2-IFNβm (Dual) and αTROP2-IFNβm (Mono) administration groups, and most indicators tended to return to the normal range over time. In addition, no significant difference in the pattern of hematological toxicity was observed between the two drugs. These results suggest that αTROP2-IFNβm (Dual) and αTROP2-IFNβm (Mono) exhibit relatively good hematological safety under a single 5 mg / kg administration condition.

[0152] <Experimental Example 4> Comparison of Pharmacokinetic Characteristics of αTROP2-IFNβm Fusion Protein in hIFNAR1 / 2 Knock-in Mice

[0153] Experiments were conducted using hIFNAR1 / 2 KI mice to compare and evaluate the pharmacokinetic parameters (PK parameters) of αTROP2-IFNβm (Dual) and αTROP2-IFNβm (Mono).

[0154] Specifically, αTROP2-IFNβm (Dual) or αTROP2-IFNβm (Mono) was administered as a single intraperitoneal (IP) dose of 5 mg / kg to healthy hIFNAR1 / 2 KI mice. Three mice were used per group, and whole blood was collected at 0, 1, 2, 4, 8, 24, 48, and 72 hours after drug administration, and plasma was separated. Drug concentrations in the separated plasma were measured using the ELISA assay. For this purpose, wash buffer (0.05% Tween-20 in 1×PBS), coating buffer (50 mM carbonate buffer, pH 9.4), and blocking buffer (1% BSA in 0.05% PBS-T) were prepared and used. 100 μL of the prepared coating solution was dispensed into 96-well plates, sealed, and incubated at 37°C for 1 hour. Subsequently, each well was washed three times with 300 μL of wash buffer. 100 μL of blocking buffer was dispensed into each well and incubated at 25°C for 1 hour, followed by three washes with wash buffer. 100 μL of the analysis sample was dispensed into each well and incubated at 25°C for 2 hours, followed by three washes with wash buffer. Next, 100 μL of the detection reagent, the Goat Anti-Human IgG light chain secondary antibody (Invitrogen, #A18853), was dispensed into each well and incubated at 25°C for 1 hour. After the incubation, the wells were washed five times with wash buffer, and 100 μL of TMB solution was dispensed into each well and incubated at 25°C for 15 minutes under light protection. The reaction was stopped with 100 μL of stop solution (Sigma, #S5814), and the absorbance was measured at 450 nm using a spectrophotometer (Tecan, Spark® Multimode Microplate Reader).

[0155] As a result, as shown in Figure 4 and Table 3, αTROP2-IFNβm (Mono) exhibited significantly improved pharmacokinetic properties compared to αTROP2-IFNβm (Dual).

[0156]

[0157] Specifically, the blood half-life (T₁ / ₂) of αTROP2-IFNβm (Mono) increased by more than six times compared to αTROP2-IFNβm (Dual), and the AUC representing total drug exposure in the body INF (h·ng / mL) increased by more than 7 times. In addition, the clearance (CL) was found to be significantly lower in αTROP2-IFNβm (Mono), confirming that it is maintained more stably in the body. Furthermore, the dose-normalized peak concentration (Cmax_D) also showed a higher value in αTROP2-IFNβm (Mono).

[0158] These results suggest that the αTROP2-IFNβm (Mono) structure, fused with one IFNβm, provides superior pharmacokinetic stability and drug exposure in vivo compared to the αTROP2-IFNβm (Dual) structure, fused with two IFNβm. These pharmacokinetic characteristics are considered to be consistent with the excellent in vivo antitumor effect observed in previous antitumor efficacy experiments.

[0159] Accordingly, αTROP2-IFNβm (Mono) was named ABN202(αTROP2) and subsequent experiments were conducted.

[0160] <Experimental Example 5> Comparative Evaluation of the In Vitro Cancer Cell Growth Inhibitory Activity of ABN202 (αTROP2)

[0161] To evaluate the in vitro anticancer activity of ABN202 (αTROP2), comparative experiments were performed with a TROP2-targeted antibody-drug conjugate (ADC) and a non-targeted interferon fusion protein.

[0162] Specifically, TROP2-targeted ADCs Trodelvy® (sacituzumab govitecan), Datopotamab deruxtecan (Dato-DXd), and Sacituzumab tirumotecan (SKB264) were used as comparative drugs, and a non-targeted interferon fusion protein Control-IFNβ mutein was included as a control.

[0163] The cell lines used in the study were two human triple-negative breast cancer (TNBC) cell lines (HCC-70, MDA-MB-231), two hormone receptor-positive breast cancer cell lines (HCC-1428, SNU-1528), and two EGFR-mutated non-small cell lung cancer (NSCLC) cell lines (HCC-827, HCC-4006 Osimertinib resistance).

[0164] Each cell line was seeded in a 96-well plate at a density of approximately 2,000–3,000 cells per well and cultured for 16 hours. Subsequently, the test drug was applied using serial dilutions starting from 100 nM. After 120 hours had passed since drug treatment, 10 μL of EZ-Cytox was added to each well and reacted for 4 hours. Cell viability was then analyzed by measuring absorbance at a wavelength of 450 nm using a spectrophotometer (Tecan, Spark® Multimode Microplate Reader).

[0165] As a result, as shown in Figures 5a to 5c, ABN202 (αTROP2) exhibited a strong cell growth inhibitory effect in triple-negative breast cancer cell lines (HCC-70, MDA-MB-231), hormone receptor-positive breast cancer cell lines (HCC-1428, SNU-1528), and EGFR-mutated non-small cell lung cancer cell lines (HCC-827, HCC-4006 Osimertinib resistance).

[0166] In particular, ABN202 (αTROP2) was confirmed to exhibit overall superior cancer cell growth inhibitory activity compared to TROP2-targeted ADCs and non-targeted interferon fusion proteins (Control-IFNβm) tested under the same conditions. These results suggest that a structure in which an IFNβ variant is fused to a TROP2-targeted antibody can induce selective anticancer activity compared to non-targeted interferons by effectively delivering interferon activity to TROP2-expressing cancer cells. Furthermore, ABN202 (αTROP2) showed a potent growth inhibitory effect not only in EGFR-mutated non-small cell lung cancer cell lines but also in osimertinib-resistant cell lines (HCC-4006 Osimertinib resistance), suggesting that it can be an effective therapeutic strategy even in cancers where drug resistance has developed. Therefore, ABN202 (αTROP2) of the present invention can be usefully utilized as a novel anticancer therapeutic agent capable of exhibiting superior anticancer activity compared to existing TROP2-targeted ADCs in various TROP2-expressing cancer types.

[0167] <Experimental Example 6> Evaluation of the in vitro anticancer activity of ABN202 (αTROP2) in ADC and chemotherapy-resistant cancer cell lines

[0168] To confirm the resistance-independent efficacy of ABN202(αTROP2), we evaluated whether ABN202(αTROP2) exhibits potent anticancer activity even in cancer cell lines resistant to existing ADCs or chemotherapy drugs.

[0169] <6-1> Construction and Characterization of HCC1954 Enhertu-Resistant Cell Line

[0170] To verify whether the ADC resistance mechanism could be evaded, the HCC1954 Enhertu-resistant cell line was constructed by treating the human breast cancer cell line HCC1954 with the HER2-targeted ADC Trastuzumab deruxtecan (Enhertu, T-DXd) for a long period and selecting cell clones (Fig. 6a).

[0171] To characterize the properties of the established resistant cell line, the expression levels of ABC transporter proteins (ABCC1, ABCG2) were analyzed at the mRNA and protein levels.

[0172] As a result, as shown in Figure 6b, it was confirmed that both ABCC1 and ABCG2 were upregulated in the HCC1954 Enhertu-resistant cell line compared to the parental cell line.

[0173] In addition, ABCC1 and ABCG2 inhibitors were treated at 10 μM and 1 μM, respectively, followed by serial dilution of T-DXd and Dato-DXd starting from 100 nM in 1 / 10 increments. After 120 hours, 10 μL of EZ-Cytox was added per well, and absorbance was measured at 450 nm after 4 hours of reaction.

[0174] As a result, as shown in Figure 6c, the HCC1954 Enhertu-resistant cell line exhibited cross-resistance to the payload (DXd) even in Dato-DXd, but it was confirmed that drug responsiveness was restored upon treatment with ABCC1 and ABCG2 inhibitors. This suggests that the constructed cell line possesses ABC transporter-dependent ADC resistance.

[0175] <6-2> Evaluation of the anticancer activity of ABN202 (αTROP2) in ADC-resistant cancer cell lines

[0176] The in vitro anticancer activity of ABN202 (αTROP2) was evaluated and compared with various ADCs using the HCC1954 Enhertu-resistant cell line.

[0177] Specifically, each cell line (parenteral, Enhertu-resistant) was seeded at a density of 3,000 cells per well in a 96-well plate, and after 16 hours, the drug was treated with serial dilutions starting from 100 nM in 1 / 10 increments. After 120 hours, 10 μL of EZ-Cytox was added, and absorbance was measured at 450 nm after 4 hours. The comparative ADCs used were Enhertu® (T-DXd), Trodelvy® (Sacituzumab govitecan), Dato-DXd (Datopotamab deruxtecan), Padcev® (Enfortumab vedotin), and SKB264 (Sacituzumab tirumotecan).

[0178] As a result, as shown in Figure 6d, most ADCs showed reduced efficacy due to cross-resistance in Enhertu-resistant cell lines, but ABN202(αTROP2) exhibited potent inhibition of cancer cell growth in both parental and Enhertu-resistant cell lines. This demonstrates that ABN202(αTROP2) is effective and unaffected by ABC transporter-dependent ADC resistance mechanisms.

[0179] <6-3> Evaluation of Anticancer Activity of ABN202 (αTROP2) in Chemotherapy-Resistant Triple-Negative Breast Cancer

[0180] The in vitro anticancer activity of ABN202 (αTROP2) was evaluated using the paclitaxel-resistant triple-negative breast cancer cell line MDA-MB-231.

[0181] Specifically, each cell line (parenteral, Paclitaxel-resistant) was seeded at a density of 3,000 cells per well in a 96-well plate, and after 16 hours, the drug was treated with serial dilutions starting from 100 nM in 1 / 10 increments. After 120 hours, 10 μL of EZ-Cytox was added, and absorbance was measured at 450 nm after 4 hours. Paclitaxel, MMAE (Monomethyl auristatin E), and Padcev® (Enfortumab vedotin) were used as control drugs.

[0182] As a result, as shown in Figure 6e, the efficacy of MMAE-based ADCs and Paclitaxel was reduced in Paclitaxel-resistant cell lines due to cross-resistance, but ABN202(αTROP2) exhibited potent inhibition of cancer cell growth in both parental and Paclitaxel-resistant cell lines. This confirms that ABN202(αTROP2) is effective even without being affected by chemotherapy resistance mechanisms.

[0183] In summary, it was confirmed that ABN202(αTROP2) exhibits potent anticancer activity even in ADC-resistant and chemotherapy-resistant cancer cell lines. This suggests the potential of ABN202(αTROP2) as a novel anticancer therapeutic agent capable of acting independently of existing ADC and chemotherapy resistance mechanisms.

[0184] <Experimental Example 7> Evaluation of In vivo Anticancer Activity of ABN202 (αTROP2) in MDA-MB-231 Triple-Negative Breast Cancer Xenograft Mouse Model

[0185] To confirm the potent tumor suppression ability and long-lasting efficacy of ABN202(αTROP2), the in vivo anticancer activity of ABN202(αTROP2) was evaluated by comparing it with existing TROP2-targeted ADCs and non-targeted interferon fusion proteins (Control-IFNβm).

[0186] Specifically, 5 × 10⁶ human triple-negative breast cancer cell lines were introduced into 7–8 week old BALB / c nude mice. 6 Cells were injected subcutaneously (SC) into the right flank. Tumor size was measured three times a week using a caliper, and the tumor size was calculated using the formula (long axis × short axis²) / 2. Mice were randomly assigned to the experimental group when the tumor size reached 100–150 mm³. The test drugs were ABN202 (αTROP2), three TROP2-targeted ADCs (Trodelvy®, Dato-DXd, SKB264), and a non-targeted IFNβm (Control-IFNβ mutein). Each drug was administered intraperitoneally (IP) at a dose of 5 mg / kg, once a week (QW) for three weeks. Tumor growth inhibition (TGI) was evaluated based on changes in tumor volume.

[0187] As a result, as shown in Fig. 7a, ABN202 (αTROP2) exhibited the best antitumor activity compared to all comparative drugs, and the tumor growth inhibition rate (TGI) was confirmed to be 92%. Notably, a complete response (CR) was observed in two mice (Fig. 7A).

[0188] In addition, the recurrence of the tumor was confirmed through long-term observation after the discontinuation of drug administration.

[0189] As a result, as shown in Figure 7b, ABN202(αTROP2) maintained a sustained anticancer effect without tumor recurrence compared to non-targeted IFNβm.

[0190] These results suggest that ABN202(αTROP2) possesses superior in vivo anticancer activity compared to TROP2-targeted ADCs and non-targeted interferons, and provides a durable tumor suppression effect.

[0191] <Experimental Example 8> Evaluation of the antitumor effect of ABN202 (αTROP2) in a PDX model derived from breast cancer patients with a history of ADC treatment

[0192] Experiments were conducted to confirm TROP2 expression in tumors derived from hormone receptor-positive (HR+) breast cancer patients with a history of ADC prescription, and to evaluate the antitumor activity of ABN202 (αTROP2) in the corresponding PDX (Patient-Derived Xenograft) mouse model.

[0193] Specifically, formalin-fixed paraffin-embedded (FFPE) blocks were prepared using tumor tissue extracted from PDX mouse models derived from HR+ breast cancer patients with a history of ADC treatment, and sections 4–5 μm thick were prepared. After deparaffinizing the FFPE slides, an antigen retrieval process was performed through heat treatment. Subsequently, non-specific binding was blocked by treatment with a blocking buffer, and an anti-TROP2 antibody (Abcam, ab214488) was incubated overnight at 4°C as the primary antibody. Next, an HRP-conjugated secondary antibody was incubated at room temperature for 1 hour, and after color development using a DAB substrate, the samples were observed under a light microscope.

[0194] As a result, as shown in Figure 8a, TROP2 expression at an IHC score of 3+ was confirmed in the ADC-resistant breast cancer PDX model.

[0195] Subsequently, the antitumor efficacy of ABN202(αTROP2) was evaluated using the same PDX model.

[0196] Specifically, PDX tumor tissue was transplanted into 7–8 week old BALB / c nude mice to induce tumor formation, and when the tumor size reached approximately 100–150 mm³, the mice were randomly assigned to an experimental group. The test substance, ABN202 (αTROP2), was administered intraperitoneally (IP) at a dose of 5 mg / kg, and the drug was administered once weekly (QW) for a total of 4 weeks.

[0197] As a result, as shown in Figure 8b, ABN202(αTROP2) was confirmed to significantly inhibit tumor growth in a PDX model derived from breast cancer patients with a history of ADC treatment. These results suggest that ABN202(αTROP2) can exhibit effective anticancer activity in tumors in which TROP2 is continuously expressed even after prior ADC treatment.

[0198] <Experimental Example 9> Evaluation of Immune Cell Activation and Anticancer Activity of ABN202 (αTROP2) in a Human PBMC Co-culture System

[0199] To determine whether ABN202(αTROP2) can enhance cancer cell death through the activation of human immune cells, co-culture experiments were performed using human peripheral blood mononuclear cells (hPBMCs) and human breast cancer cell lines.

[0200] Specifically, concentrated peripheral blood obtained from the LRS chamber was diluted with 2% FBS / PBS. Subsequently, 25 mL of Histopaque®-1077 (Sigma) was dispensed into a 50 mL tube, and 25 mL of the diluted peripheral blood was slowly added to form a layer. The tube was then centrifuged at 1200 g for 10 minutes. After centrifugation, the supernatant was removed, and the human peripheral blood mononuclear cell (PBMC) layer was recovered and transferred to a new 50 mL tube. The PBMCs were then obtained by washing three times at 300 g for 8 minutes each with 2% FBS / PBS washing buffer. Finally, human breast cancer cell line ZR-75-1 was seeded at 5,000 cells per well and PBMCs at 10,000 cells per well (Effective : Target ratio = 2 : 1) in a 96-well plate for co-culture. Cancer cell growth inhibitory ability was evaluated after 72 hours of co-culture, and immune cell activation marker analysis was performed after 24 hours of co-culture (Fig. 9a).

[0201] As a result, as shown in Figure 9b, while there was no significant difference in cancer cell killing ability of TROP2-targeted ADCs depending on whether they were co-cultured with PBMCs, it was confirmed that ABN202 (αTROP2) showed a significant increase in cancer cell killing ability under co-culture conditions with PBMCs. This suggests that ABN202 (αTROP2) can enhance anticancer activity through the activation of immune cells.

[0202] In addition, flow cytometry was performed to confirm whether PBMC was activated after 24 hours of co-culture.

[0203] Specifically, PBMCs were recovered from the supernatant of the co-culture medium and seeded at a density of 5,000 cells in 100 μL of 1% FBS / PBS. Subsequently, 1 μL each of human CD44, CD62L, and CD69 antibodies were added and incubated on ice for 1 hour, followed by washing three times with 1% FBS / PBS. Afterward, the expression of PBMC activation markers was analyzed using a flow cytometer (BD FACS Lyric).

[0204] As a result, as shown in Fig. 9c, unlike TROP2-targeted ADCs, ABN202(αTROP2) was found to significantly increase the expression of CD69, a human immune cell activation marker, under PBMC co-culture conditions. These results suggest that ABN202(αTROP2) can induce anticancer effects not only through a simple cytotoxic mechanism but also through immune cell activation.

[0205] <Experimental Example 10> Analysis of the antitumor effect of ABN202 (αTROP2) in the hTROP2-MB49 model for evaluation of immune cell-dependent anticancer activity

[0206] In order to confirm whether the anticancer activity of ABN202 (αTROP2) can be expressed not only through a direct cancer cell death effect but also through an immune cell-mediated anticancer effect, in vitro and in vivo experiments were performed using a mouse MB49 cell line (hTROP2-MB49) overexpressing human TROP2.

[0207] Specifically, the in vitro cancer cell growth inhibitory activity of ABN202 (αTROP2) against the hTROP2-MB49 cell line was evaluated by comparison with TROP2-targeted ADCs. Trodelvy® (Sacituzumab govitecan), Datopotamab deruxtecan (Dato-DXd), and Sacituzumab tirumotecan (SKB264) were used as comparator drugs. 2,000 cells per well were seeded in a 96-well plate, and after 16 hours, the test drugs were treated by serial dilution starting from 100 nM in 1 / 10 increments. 120 hours after drug treatment, 10 μL of EZ-Cytox was added per well, and after 4 hours, the absorbance was measured at 450 nm using a spectrophotometer (Tecan, Spark® Multimode Microplate Reader).

[0208] As a result, as shown in Figure 10a, since the hTROP2-MB49 cell line does not express human interferon alpha receptor (IFNAR), ABN202 (αTROP2) did not induce direct cancer cell death in the cell line, whereas TROP2-targeted ADCs were found to induce potent cancer cell death.

[0209] Next, to evaluate the immune cell-mediated anticancer activity of ABN202 (αTROP2), splenocytes were isolated from hIFNAR1 / 2 knock-in mice and co-cultured with the hTROP2-MB49 cell line. For this purpose, the spleens of healthy hIFNAR1 / 2 KI mice were immersed in 1% FBS / PBS and mechanically pulverized on a 70 μm strainer (Corning, #431751) to isolate the cells. After centrifuging the isolated tissue, red blood cells were removed by treatment with RBC lysis buffer (Invitrogen, #00-4333-57), and splenocytes were obtained through a washing process. Subsequently, 5,000 cells of the hTROP2-MB49 cell line and 10,000 cells of splenocytes were seeded into a 96-well plate (Effective : Target ratio = 4 : 1) and co-cultured for 72 hours. ABN202(αTROP2) was treated with serial dilution starting from 100 nM in 1 / 10 increments, and EZ-Cytox analysis was performed after 72 hours.

[0210] As a result, as shown in Figure 10b, it was confirmed that ABN202(αTROP2) significantly increased the cancer cell killing effect under co-culture conditions with splenocytes derived from hIFNAR1 / 2 KI mice.

[0211] In addition, hTROP2-MB49 cell line was injected into hIFNAR1 / 2 KI mice at a rate of 1 × 10⁶ per mouse. 6Tumors were formed by injecting cells one by one subcutaneously (SC) into the right flank, and in vivo anticancer activity was evaluated. When the tumor size reached approximately 100–150 mm³, mice were randomly assigned to the experimental group, and the test drug was administered intraperitoneally (IP) at a dose of 5 mg / kg. The drug was administered once a week (QW) for a total of 3 weeks. Trodelvy® (Sacituzumab govitecan), Datopotamab deruxtecan (Dato-DXd), and Sacituzumab tirumotecan (SKB264) were used as comparator drugs.

[0212] As a result, as shown in Fig. 10c, although no direct apoptotic effect was observed in the hTROP2-MB49 cell line (Fig. 10a), ABN202 (αTROP2) exhibited superior in vivo antitumor activity compared to TROP2-targeted ADCs through immune cell-mediated anticancer effects. These results suggest that ABN202 (αTROP2) can exert a potent antitumor effect through an anticancer mechanism involving immune cell activation.

[0213] <Experimental Example 11> Evaluation of Tumor-Targeted Immuno-Anticancer Effects of ABN202(αTROP2) in hIFNAR1 / 2 KI Mouse Tumor Model

[0214] To determine whether the in vivo immuno-oncology activity of ABN202 (αTROP2) is enhanced compared to treatment with antibody or interferon alone, its antitumor efficacy was evaluated in a tumor model using hIFNAR1 / 2 knock-in mice.

[0215] <11-1> Evaluation of the antitumor activity of ABN202 (αTROP2) in the hTROP2-B16F10 tumor model

[0216] Specifically, B16F10 cell lines overexpressing human TROP2 (hTROP2-B16F10) were injected into hIFNAR1 / 2 KI mice at a rate of 1 × 10 per mouse. 6Tumors were induced by injecting cells subcutaneously (SC) into the right flank. Tumor size was measured three times a week using a caliper, and tumor volume was calculated using the formula (long axis × short axis²) / 2. When the tumor size reached approximately 100–150 mm³, mice were randomly assigned to experimental groups and administered drugs. The test drugs consisted of groups treated with ABN202 (αTROP2), a non-target antibody (Control mAb), a TROP2 target antibody (Sacituzumab), and IFNβm alone. Each drug was administered intraperitoneally (IP) at a dose of 10 mg / kg, three times a week (TIW) for a total of two weeks.

[0217] As a result, as shown in Figure 11a, ABN202(αTROP2) exhibited a superior tumor growth inhibitory effect compared to the groups treated with non-targeted antibodies and TROP2 targeting antibodies alone, as well as the group treated with IFNβm alone. These results suggest that the fusion structure of the TROP2 targeting antibody and the IFN-β variant can induce enhanced anti-tumor activity compared to when each is used alone.

[0218] <11-2> Evaluation of Tumor-Targeted Immuno-Anticancer Activity of ABN202 (αTROP2) in hTROP2-MB49 Tumor Model

[0219] In addition, in vivo experiments using an hTROP2-MB49 tumor model were performed to confirm the tumor-targeted immuno-anticancer effect of ABN202 (αTROP2).

[0220] Specifically, the MB49 cell line (hTROP2-MB49) overexpressing human TROP2 was injected into hIFNAR1 / 2 KI mice at a rate of 1 × 10⁶ per mouse. 6Tumors were formed by injecting cells one by one subcutaneously (SC) into the right flank. Mice were randomly assigned to the experimental group when the tumor size reached approximately 100–150 mm³. The test drugs were ABN202 (αTROP2) and non-targeted interferon fusion protein (Control-IFNβm), and the drugs were administered intraperitoneally (IP) at a dose of 1 mg / kg. The drugs were administered once a week (QW) for a total of 2 weeks.

[0221] As a result, as shown in Figure 11b, ABN202 (αTROP2) exhibited a superior tumor growth inhibitory effect through tumor-targeted interferon delivery compared to the non-targeted interferon fusion protein (Control-IFNβm) with an improved half-life. These results suggest that fusion of an IFN-β variant with a TROP2-targeted antibody enables selective delivery to tumor tissue, thereby inducing an enhanced immuno-oncology effect.

[0222] <Experimental Example 12> Evaluation of Dose-Dependent Antitumor Activity of ABN202 (αTROP2) in hTROP2-MB49 Tumor Model

[0223] To determine whether the in vivo antitumor activity of ABN202 (αTROP2) is dose-dependent, experiments were conducted to evaluate the dose-response relationship in a tumor model using hIFNAR1 / 2 knock-in mice.

[0224] Specifically, the MB49 cell line (hTROP2-MB49) overexpressing human TROP2 was injected into hIFNAR1 / 2 KI mice at a rate of 1 × 10⁶ per mouse. 6Tumors were formed by injecting cells subcutaneously (SC) into the right flank. Tumor size was measured three times a week using a caliper, and tumor volume was calculated using the formula (long axis × short axis²) / 2. When the tumor size reached approximately 100–150 mm³, mice were randomly assigned to the experimental group, and the test drug ABN202 (αTROP2) was administered intraperitoneally (IP) at doses of 5 mg / kg, 2.5 mg / kg, 1.25 mg / kg, and 0.625 mg / kg. The drug was administered once a week (QW) for a total of three weeks.

[0225] As a result, as shown in Figure 12, ABN202(αTROP2) was confirmed to exhibit dose-dependent antitumor activity, with the tumor growth inhibitory effect gradually increasing as the administered dose increases. These results suggest that ABN202(αTROP2) exhibits stable pharmacological activity in vivo and possesses the characteristic of systematically increasing antitumor efficacy with increasing dose.

[0226] <Experimental Example 13> Evaluation of Antitumor Activity of ADC and Immune Checkpoint Inhibitor Combination Therapy Group and ABN202 (αTROP2) Monotherapy Group

[0227] To evaluate the in vivo immuno-oncology activity of ABN202 (αTROP2) compared with existing TROP2-targeted ADCs and immune checkpoint inhibitors, antitumor efficacy was analyzed in a tumor model using hIFNAR1 / 2 knock-in mice.

[0228] Specifically, 1 × 10 PyMT-N cell line overexpressing human TROP2 in hIFNAR1 / 2 KI mice per mouse 6Tumors were formed by injecting cells subcutaneously (SC) into the right flank. Tumor size was measured three times a week using a caliper, and tumor volume was calculated using the formula (long axis × short axis²) / 2. Mice were randomly assigned to the experimental group when the tumor size reached approximately 100–150 mm³. The test drugs, ABN202 (αTROP2) and the TROP2-targeted ADC Trodelvy, were administered intraperitoneally (IP) at a dose of 5 mg / kg, respectively. Additionally, an anti-mouse PD-1 antibody (BioXcell, #BE0146) was used as an immune checkpoint inhibitor and administered three times weekly (TIW) at a dose of 10 mg / kg for a total of three weeks.

[0229] As a result, as shown in Figure 13a, it was confirmed that ABN202 (αTROP2) exhibited a superior tumor growth inhibitory effect compared not only to the TROP2-targeted ADC monotherapy group and the PD-1 antibody monotherapy group, but also to the combination therapy group of an ADC and an immune checkpoint inhibitor. These results suggest that ABN202 (αTROP2) may exhibit enhanced immuno-oncology activity compared to existing antibody-drug conjugate or immune checkpoint inhibitor-based treatment strategies.

[0230] <Experimental Example 14> Evaluation of Receptor Binding and Cell Signaling Activities of IFNβm and ABN2O2 (αTROP2) Under Acidic Conditions Mimicking the Tumor Microenvironment

[0231] The tumor microenvironment is known to be acidic (pH approximately 6.0–6.8) compared to normal tissue. Accordingly, to confirm changes in the receptor binding ability and cell signaling activity of interferon under acidic conditions, experiments were conducted to compare IFNα2b and IFNβm.

[0232] <14-1> Confirmation of Interferon Receptor Binding Ability of IFNα2b and IFNβm Under Acidic Conditions

[0233] First, the interferon receptor binding ability in an acidic environment was evaluated using the ELISA assay.

[0234] Specifically, IFNα2b (PBL #11105) and IFNβm were diluted to a concentration of 100 nM in PBS buffer and dispensed in 100 μL aliquots onto 96-well ELISA plates (Corning #2592), followed by coating at 4°C for 16 hours. Subsequently, all buffers except the coating buffer were adjusted using 1N HCl from pH 7.2 to pH 5.0 in increments of 0.2. After washing the coated plates three times with washing buffer (0.05% PBS-T, pH 7.4 or pH 6.0), 300 μL of blocking buffer (50 mg / mL BSA in PBS, pH 7.2–5.0) was added, and the plates were reacted at room temperature for 1 hour. Subsequently, the IFNAR1 / 2 fusion protein was serially diluted in 1 / 3 increments starting from 100 nM in reagent diluent (1 mg / mL BSA in 0.05% PBS-T, pH 7.2–5.0), and 100 μL was added to each well and incubated at room temperature for 1 hour. After washing, 100 μL of anti-human IgG-HRP antibody (Jackson Lab #109-035-003) diluted 1:5000 was added and incubated for 1 hour. Afterward, 100 μL of TMB substrate (Surmodics #TMBW 1000-01) was added, and the absorbance was measured at 450 nm after a 20-minute reaction.

[0235] As a result, as shown in Figure 14a, the binding ability of IFNα2b to the IFNAR receptor decreased as the pH decreased, whereas IFNβm showed a tendency to increase its binding ability to the receptor as the pH decreased.

[0236] <14-2> Confirmation of Cell Signaling Ability of IFNα2β and IFNβm under Acidic Conditions

[0237] Next, experiments using Daudi cells were conducted to evaluate the cell signaling activity of interferon in an acidic environment.

[0238] Specifically, 2 × 10 Daudi cells in a 6-well plate 6 Cells were seeded aliquots and cultured in neutral medium (pH 7.4) or acidic medium adjusted to pH 6.5 using 1N HCl. Subsequently, IFNα2b and IFNβm were treated at concentrations of 10 nM, 1 nM, and 0.1 nM, respectively, for 24 hours. After treatment, the cells were harvested, and proteins were extracted by adding RIPA buffer, a protease inhibitor, and a phosphatase inhibitor. The proteins were then separated by SDS-PAGE, transferred to a PVDF membrane, and subjected to Western blot analysis using the anti-human pSTAT-1 antibody (Cell Signaling Technology).

[0239] As a result, as shown in Figure 14b, IFNα2b reduced the STAT1 phosphorylation (pSTAT-1) signal in an acidic environment, whereas IFNβm actually increased signal transduction activity in an acidic environment, confirming that the pSTAT-1 signal was activated even at a concentration more than 10 times lower than under neutral conditions.

[0240] <14-3> Confirmation of Cell Signaling Ability of αTROP2-IFNα2b and ABN202(αTROP2) under Acidic Conditions

[0241] In addition, the cell signaling activity of αTROP2-IFNα2b and ABN202 (αTROP2) was compared and evaluated according to pH conditions using the same method. The sequence of αTROP2-IFNα2b is shown in Table 4 below and was produced through gene introduction and expression purification as described in Example 1 above.

[0242]

[0243] As a result, as shown in Fig. 14c, the signaling activity of αTROP2-IFNα2b was reduced in an acidic environment, whereas ABN202(αTROP2) was found to activate pSTAT-1 signaling in an acidic environment even at concentrations more than 10 times lower than in a neutral environment. In summary, this suggests that IFNβm and ABN202(αTROP2) containing it may exhibit enhanced receptor binding and signaling activity under acidic conditions similar to the tumor microenvironment, demonstrating potential advantages in inducing tumor tissue-specific immune activation.

[0244] <Experimental Example 15> Confirmation of Antitumor Efficacy of ABN202 (αTROP2) Compared to Type 1 Interferon-Based Immunocytokine in hTROP2-MB49 Tumor Model

[0245] To evaluate the in vivo immuno-anticancer activity of ABN202 (αTROP2) compared with αTROP2-IFNβ and αTROP2-IFNα2b, the anti-tumor efficacy was analyzed in a tumor model using hIFNAR1 / 2 knock-in mice.

[0246] Specifically, the MB49 cell line (hTROP2-MB49) overexpressing human TROP2 was injected into hIFNAR1 / 2 KI mice at a rate of 1 × 10⁶ per mouse. 6Tumors were induced by injecting cells subcutaneously (SC) into the right flank. Tumor size was measured three times a week using a caliper, and tumor volume was calculated using the formula (long axis × short axis²) / 2. When the tumor size reached approximately 100–150 mm³, mice were randomly assigned to an experimental group and administered the test drugs. The test drugs were ABN202 (αTROP2), αTROP2-IFNβ, and αTROP2-IFNα2b, each administered intraperitoneally (IP) at a dose of 5 mg / kg. The drugs were administered once a week (QW) for a total of three weeks.

[0247] As a result, as shown in Figure 15, ABN202(αTROP2) exhibited the most superior tumor growth inhibitory effect compared to the control drugs αTROP2-IFNβ and αTROP2-IFNα2b. In particular, complete response (CR) was observed in 5 out of 6 mice in the ABN202(αTROP2) treatment group. These results suggest that ABN202(αTROP2) may exhibit enhanced immuno-oncology activity compared to existing interferon-based antibody fusion proteins.

[0248] <Experimental Example 16> Analysis of the Antitumor Mechanism of ABN202 (αTROP2) through Immune Cell-Dependent and Tumor Microenvironment Immunomodulatory Effects

[0249] Experiments were conducted using an hIFNAR1 / 2 knock-in mouse tumor model to determine whether the immuno-oncology effect of ABN202 (αTROP2) is expressed in a manner dependent on specific immune cells, and to analyze changes in immune cells within the tumor microenvironment and tumor-draining lymph nodes (TDLNs).

[0250] <16-1> Confirmation of Immunocyte-Dependent Antitumor Effect of ABN202(αTROP2)

[0251] First, immune cell depletion experiments were performed to determine whether the antitumor effect of ABN202 (αTROP2) depends on specific immune cells. Specifically, the MB49 cell line (hTROP2-MB49) overexpressing human TROP2 was introduced into hIFNAR1 / 2 KI mice at a rate of 1 × 10⁶ per mouse. 6 Tumors were induced by injecting cells subcutaneously (SC) into the right flank. Tumor size was measured three times a week using a caliper, and tumor volume was calculated using the formula (long axis × short axis²) / 2. Mice were randomly assigned to the experimental group when the tumor size reached approximately 100–150 mm³. Subsequently, ABN202 (αTROP2) was administered intraperitoneally (IP) at a dose of 5 mg / kg after specific immune cells had been selectively eliminated using CD4, CD8, CD11c, CSF1R, and NK1.1 depletion antibodies (BioXcell) targeting respective immune cell markers. The drug was administered once a week (QW) for a total of three weeks.

[0252] As a result, as shown in Figure 16a, the antitumor effect of ABN202 (αTROP2) was significantly reduced under conditions where CD8 T cells were removed. On the other hand, under conditions where CD4 T cells or NK cells were removed, a tendency for the antitumor effect to increase was observed. These results suggest that the antitumor immune effect of ABN202 (αTROP2) is expressed in a CD8 T cell-dependent manner.

[0253] <16-2> Analysis of Immune Cell Changes in the Tumor Microenvironment Caused by ABN202 (αTROP2)

[0254] Next, the effect of ABN202(αTROP2) on immune cell composition within the tumor microenvironment was analyzed.

[0255] To this end, hTROP2-MB49 cell lines were transplanted into hIFNAR1 / 2 KI mice in the same manner to induce tumor formation. When the tumor size reached approximately 200–300 mm³, ABN202 (αTROP2) was administered once intraperitoneally (IP) at a dose of 5 mg / kg. Five days after drug administration, tumor tissue was isolated and separated into single cells using a Tumor Dissociation Kit (Miltenyi Biotec, #130-096-730). Subsequently, antibodies against cell surface markers were applied, and the composition of immune cells within the tumor was analyzed using a flow cytometer (BD FACS Lyric).

[0256] As a result, as shown in Figure 16b, it was confirmed that the proportion of effector CD8 T cells within the tumor increased and the proportion of G-MDSCs (granulocytic myeloid-derived suppressor cells), known as immunosuppressive cells, decreased in the ABN202 (αTROP2) treatment group.

[0257] <16-3> Analysis of Immune Cell Changes in Tumor Drainage Lymph Nodes Caused by ABN202 (αTROP2)

[0258] In addition, the effect of ABN202 (αTROP2) on the immune response in tumor-draining lymph nodes (TDLNs) was analyzed.

[0259] To this end, ABN202 (αTROP2) was administered once intraperitoneally (IP) at a dose of 5 mg / kg to hIFNAR1 / 2 KI mice that had formed hTROP2-MB49 tumors. Tumor drainage lymph nodes were isolated at 1, 3, and 5 days after drug administration. Single-cell suspensions were prepared by mechanically separating the isolated lymph node tissues in a 1% FBS / PBS solution using a 70 μm strainer (Corning #431751). Subsequently, the suspensions were treated with cell surface marker antibodies and tumor-specific peptide-tetramers, and then analyzed using a flow cytometer (BD FACS Lyric).

[0260] As a result, as shown in Fig. 16c, it was confirmed that ABN202(αTROP2) increased the ratio of tumor-specific CD8 T cells and dendritic cell subtypes over time (Fig. 16C).

[0261] In summary, this suggests that ABN202(αTROP2) can activate a CD8 T cell-dependent anti-tumor immune response by alleviating the immunosuppressive environment in the tumor microenvironment and inducing a tumor-specific T cell response.

[0262] <Experimental Example 17> Analysis of Long-Term Anti-Tumor Memory Immunity and CD8 T Cell-Mediated Immune Transfusion Effects Induced by ABN202 (αTROP2)

[0263] To determine whether the anti-tumor immune response induced by ABN202 (αTROP2) is maintained as long-term memory immunity and whether the immune response is mediated by CD8 T cells, rechallenge experiments and adaptive cell transfer (ACT) experiments were performed.

[0264] <17-1> Long-term anti-tumor memory immunity and rechallenge experiment with ABN202(αTROP2)

[0265] First, we evaluated whether long-term anti-tumor memory immunity was formed in mice that achieved complete remission induced by ABN202 (αTROP2).

[0266] To this end, the MB49 cell line (hTROP2-MB49) overexpressing human TROP2 was injected into hIFNAR1 / 2 KI mice at a rate of 1 × 10⁶ per mouse. 6 Tumors were formed by injecting cells subcutaneously (SC) into the right flank. Tumor size was measured three times a week using a caliper, and tumor volume was calculated using the formula (long axis × short axis²) / 2. When the tumor size reached approximately 100–150 mm³, mice were randomly assigned to an experimental group, and ABN202 (αTROP2) was administered intraperitoneally (IP) at a dose of 5 mg / kg. The drug was administered once a week (QW) for a total of three weeks. Subsequently, mice that achieved a complete response (CR) to ABN202 (αTROP2) treatment were observed for an additional 55 days, confirming that no tumor recurrence occurred.

[0267] To confirm the presence of anti-tumor memory immunity in mice in long-term remission, the same hTROP2-MB49 cell line was used at a rate of 1 × 10⁶ per mouse. 6 Tumor rechallenge was performed by injecting cells one by one subcutaneously (SC) into the opposite flank (left flank). As controls, hIFNAR1 / 2 KI normal mice of similar age and mice showing a partial response (PR) were used.

[0268] As a result, as shown in Figure 17a, it was confirmed that complete remission was maintained without tumor formation in 3 out of 5 mice treated with ABN202 (αTROP2) even in response to tumor re-challenge. These results suggest that ABN202 (αTROP2) can induce long-lasting anti-tumor memory immunity.

[0269] <17-2> Evaluation of CD8 T cell-mediated anti-tumor immune transfer (adoptive transfer)

[0270] Next, to determine whether memory immunity formed by ABN202 (αTROP2) is mediated by CD8 T cells, an adaptive cell transfer (ACT) experiment was performed.

[0271] To this end, spleens were isolated from mice that achieved complete remission (CR) or partial remission (PR) upon treatment with ABN202 (αTROP2), as well as from normal hIFNAR1 / 2 KI mice of similar age that had not been administered the drug (aged healthy) and tumor-experienced mice. After isolating CD8 T cells from the isolated splenocytes, 5 × 10⁶ cells were administered to recipient mice per mouse. 5 Adoptive cell transfer was performed by injecting individual cells intravenously (IV) through the tail vein. Subsequently, changes in tumor growth were observed.

[0272] As a result, as shown in Figure 17b, it was confirmed that tumor growth was significantly inhibited in the experimental group that received CD8 T cells derived from mice that achieved complete or partial remission by ABN202 (αTROP2) treatment.

[0273] In summary, ABN202(αTROP2) can induce long-lasting anti-tumor memory immunity, suggesting that this immune response is mediated by CD8 T cells. Furthermore, it demonstrates that this anti-tumor memory immunity can be transferred to other individuals via CD8 T cells.

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-TROP2 (Trophoblast cell surface antigen 2) 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. 16, 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.

17. Fusion protein.

10. In Paragraph 1, The above anti-TROP2 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.

11. In Paragraph 1, Characterized that the above antibody is sacituzumab or datopotamab, Fusion protein.

12. A polynucleotide encoding a fusion protein of any one of claims 1 to 11.

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 breast cancer, triple-negative breast cancer, colorectal cancer, colorectal cancer, head and neck cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, stomach cancer, pancreatic cancer, liver cancer, biliary tract cancer, gallbladder cancer, esophageal cancer, kidney cancer, bladder cancer, ureteral cancer, urothelial carcinoma, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine cancer, testicular cancer, thyroid cancer, parathyroid cancer, skin cancer, melanoma, brain cancer, brain and spinal cord tumor, thymoma, mesothelioma, lymphoma, blood cancer, acute leukemia, chronic leukemia, multiple myeloma, myelodysplastic syndromes (MDS), myelofibrosis, endocrine cancer, and sarcoma. Pharmaceutical composition for the prevention or treatment of cancer.

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

Images