Composition and method for treating cancer and fibrotic diseases

Abstract

The present invention relates to antibodies (e.g., bispecific antibodies or antigen-binding fragments thereof) and compositions containing the same. Furthermore, the present invention relates to polynucleotides encoding antibodies or antigen-binding fragments thereof, vectors containing the same, and host cells. Furthermore, the present invention relates to a method for inhibiting the binding of antibodies or antigen-binding fragments thereof to fibronectin in cells and a method for treating cancer and fibrotic diseases. Furthermore, the present invention relates to simultaneously inhibiting the binding of ligands to specific combinations of integrin receptors. Furthermore, the present invention relates to inhibiting the expression of predetermined combinations of integrin receptor subunits through the action of nucleic acids on mRNA.

Description

Technology Field

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 619,144 filed January 9, 2014, the entire contents of which are incorporated herein by reference.

[0002] The present invention was made with government support under Project Nos. CA220512, T32IR4672, T29FT0343, and K01OD030513 awarded by the National Institutes of Health. The government holds certain rights to the present invention.

[0003] Submitted together with the present application 1548-7WO_ST26.xml An XML-formatted sequence listing named is 17,519 bytes It has the size of, December 24, 2024 It has been created, and the entire disclosure thereof is incorporated herein by reference.

[0004] The present invention relates to an antibody (e.g., a bispecific antibody) or an antigen-binding fragment thereof, and a composition comprising the same. Furthermore, the present invention relates to a polynucleotide encoding said antibody or its antigen-binding fragment, and a vector comprising the same. Furthermore, the present invention relates to a method of using an antibody or its antigen-binding fragment to prevent cells from binding to fibronectin, and a method of treating fibrotic diseases and cancer. Furthermore, the present invention relates to simultaneously inhibiting the binding of a ligand to a specific combination of integrin receptors. Furthermore, the present invention relates to inhibiting the expression of a predetermined combination of integrin receptor subunits through the action of a nucleic acid on mRNA. Background Technology

[0005] Fibrosis, or scarring, occurs due to the excessive and uncontrolled accumulation of extracellular matrix (ECM) components. Strategies to limit fibrosis can be applied to a wide variety of diseases. When fibrosis progresses to a high degree, it can eventually lead to organ dysfunction and death. Fibrosis is a pathological feature observed in most chronic inflammatory diseases. Fibrosis is also a critical component of the tumor microenvironment that supports tumor progression and enables metastatic spread.

[0006] One of the major mediators of fibrosis is fibronectin, a large glycoprotein capable of influencing various key cellular functions. While fibronectin plays a crucial role in wound healing and embryonic development, it is also associated with various pathological conditions, including cancer and fibrosis. Fibronectin acts as a transient matrix protein that functions as a scaffold for the deposition of other extracellular matrix (ECM) components, such as collagen, proteoglycans, and growth factors, ultimately regulating the formation and structure of the ECM. For example, fibronectin is essential for the polymerization of type I collagen and the assembly of fibrils by fibroblasts (Sottile et al. (2002) Mol. Biol. Cell 13(10):3546-3559; Shi et al. (2010) Am. J. Physiol. Cell Physiol. 298(5):C1265-C1275), cancer-associated fibroblasts promote the directional migration of cancer cells by aligning fibronectin (Erdogan et al. (2017) J. Cell Biol. 216:3799-3816). Therefore, interfering with the function of fibronectin may provide an opportunity to destabilize or impair the function of fibronectin scaffolds that are important for the formation of additional extracellular matrix components that contribute to the progression of various diseases, including cancer.

[0007] Cells interact with fibronectin using specialized cell surface receptors, such as integrins and proteoglycans like syndecans. Integrins are expressed in the cells of most multicellular animals, and most adult mammals constitutively express various types of different integrins. Integrins possess a large extracellular domain for ligand binding, and numerous α and β subunits have been identified; these subunits combine to form different integrin heterodimers. Integrins are composed of α and β subunits, which form at least 24 different heterodimeric cell surface receptor families, which may have overlapping or unique physiological functions. Integrins α5β1 and αvβ3 are the receptors that cells primarily use to bind to fibronectin, and some additional integrins, including αvβ1, αvβ6, αvβ8, α4β1, α4β7, and α9β1, may also be involved in this binding in some cell types.

[0008] The progression of many pathological conditions, including fibrotic diseases and cancer, is accelerated when various cell types produce and / or respond to fibronectin. Fibroblasts are stromal cells that, upon activation, produce fibronectin molecules and secrete them into their surrounding environment. Integrins present on the surface of fibroblasts bind to these "globular" fibronectin molecules and anchor them, enabling the formation of larger "fibrillar" fibronectin polymers. Fibrillar fibronectin acts as a scaffold to support and anchor additional extracellular matrix proteins, such as collagen and proteoglycans. If fibronectin production is not strictly regulated, it can lead to the formation of a fibrotic environment characterized by increased matrix stiffness and the capture of activating factors, which promotes and exacerbates various disease states, such as fibrotic diseases and cancer. Preventing fibroblasts from interacting with fibronectin using integrins inhibits the formation of fibrous fibronectin polymers, thereby providing an opportunity to halt this series of processes leading to fibrosis.

[0009] In addition to fibroblasts, other cell types can interact with fibronectin and associated extracellular matrix proteins using integrins. For example, preventing tumor cells from interacting with fibronectin can prevent them from reprogramming into a more aggressive and stem-cell-like state. Similarly, preventing endothelial cells from binding to fibronectin can inhibit the proliferation and invasion of endothelial cells that mediate pathological angiogenesis in various environments. Therefore, agents capable of inhibiting the binding of fibronectin receptors to fibronectin have the potential to provide therapeutic value for various diseases, including cancer and fibrosis.

[0010] Antibodies are proteins that bind to specific antigens. The binding of antibodies to antigens selectively expressed in cells can exert antifibrotic or antitumor effects by directly blocking the function of the antigens that promote the growth or survival pathways of tumor cells. As previously mentioned, preventing various cell types from interacting with fibronectin can provide therapeutic benefits. In the case of cancer, antibodies may act as a bridge connecting tumor cells and immune effector cells, thereby indirectly inducing the destruction of tumor cells by immune effector cells. This "antibody-dependent" mode of tumor cell death mediated by immune effector cells can act additively or synergistically with direct blocking activity that inhibits the biological reprogramming of tumor cells by preventing cells from interacting with fibronectin. In this context, antibodies targeting fibronectin-binding integrins may possess dual and complementary mechanisms of action, including activity against various cell types involved in the initiation and progression of cancer.

[0011] Recently, a bispecific antibody targeting not only α5β1 but all αv integrins has been developed, exhibiting improved properties compared to monospecific antibodies in prostate cancer cells (Joshi et al. (2020) Mol. Cancer Res. 18(1):27-32). Additional bispecific antibodies have also been disclosed (Liu et al. (2015) Mol. Pharm. 12(7):2544-2550; Shlamkovich et al. (2018) BMC Biol. 16:92; Gallo et al. (2021) J. Mol. Biol. 443:15; International Patent Publication WO 2019 / 089544 A1; Joshi et al. (2019) Cancer Res. 79(13_Suppl.):902; Bhatta et al. (2021) MAbs.13(1):1859049). However, there is still a need for new compositions and methods of using such compositions to treat, for example, fibrotic diseases and to prevent cells from interacting with fibronectin. means of solving the problem

[0012] The present invention is based on an understanding of the mechanism by which fibroblasts utilize integrins to form fibrous fibronectin scaffolds that support the formation of more complex extracellular matrices, as well as the mechanism by which various cell types benefit from interactions with fibronectin. It is important to specifically target these interactions. This is because an antibody that simply recognizes only an integrin subunit (e.g., alpha-v) constituting a single possible fibronectin receptor can abnormally inhibit the function of many normal cells containing that integrin.

[0013] Since the formation of integrin heterodimers depends on the presence of both subunits, α5β1, αvβ3, and αvβ6, which are heterodimers that bind to fibronectin, are each expressed more restrictively in normal inactive epithelium. Therefore, these integrin heterodimers are low in normal adult tissues and are selectively abundant as epithelial tumors reach more aggressive and advanced terminal stages and acquire drug resistance, making them more desirable as cellular antigens for targeted therapy.

[0014] Another function of therapeutic antibodies is to induce effector cells to kill tumors or to induce apoptosis in tumor cells by conjugating them to drugs. By using antibodies that target mesenchymal-like integrins, such as β3, α5, and β6, which are selectively abundant in macrophages, there is a possibility of achieving tumor-killing effects with excellent tumor selectivity. Since specific tumors may require different effector cells, a system capable of flexibly selecting integrin antigens along with various antibody-mediated death mechanisms is advantageous. For example, this includes inducing immune effector cells to effectively mediate antibody-dependent cytotoxicity (ADCC) against epithelial cancer cells, and / or having antibodies interact with macrophages to promote antibody-dependent phagocytosis (ADCP) by utilizing macrophages as effector cells.

[0015] Without being bound by theory, inhibiting the binding of fibronectin to one or more integrins using a single therapeutic agent can provide a superior therapeutic range across heterogeneous cell populations when antigens are recognized in different tumor cells, and can provide superior recognition, apoptosis, and / or inhibition when both antigens are recognized in a single tumor cell, thereby more effectively enhancing targeting to the tumor, and / or can inhibit binding to both spherical and fibrous forms of fibronectin. In one embodiment, inhibiting cell binding to fibronectin by simultaneously interfering with the interaction between fibronectin and two integrins, for example, α5β1 and αvβ3, can more effectively block binding and thereby inhibit further progression of cancer or fibrotic disease.

[0016] Accordingly, one aspect of the present invention relates to a bispecific antibody or an antigen-binding fragment thereof that binds to two different integrins, α5β1 and αvβ3.

[0017] One aspect of the present invention relates to a bispecific antibody or an antigen-binding fragment thereof comprising a first variable domain specifically binding to integrin αvβ3 and a second variable domain specifically binding to integrin α5β1, and a composition or pharmaceutical composition comprising said bispecific antibody or the antigen-binding fragment thereof.

[0018] Another aspect of the present invention relates to a polynucleotide encoding a bispecific antibody of the present invention or an antigen-binding fragment thereof, a vector comprising said polynucleotide, and a host cell.

[0019] Another aspect of the present invention relates to a method for inhibiting the binding of cells to fibronectin, comprising the step of contacting cells with an effective amount of an antibody or an antigen-binding fragment thereof that targets one or more fibronectin-binding integrins (e.g., a bispecific antibody or an antigen-binding fragment thereof).

[0020] Another aspect of the present invention relates to a method for treating cancer in a subject requiring treatment for cancer expressing integrin αvβ3 and integrin α5β1, comprising the step of administering to the subject an antibody targeting one or more fibronectin-binding integrins or an antigen-binding fragment thereof (e.g., a bispecific antibody or an antigen-binding fragment thereof), or a therapeutically effective amount of a pharmaceutical composition of the present invention, thereby treating the cancer.

[0021] Another aspect of the present invention relates to a method for treating epithelial cell carcinoma in a subject requiring treatment for epithelial cell carcinoma, comprising the step of administering to the subject an antibody targeting one or more fibronectin-binding integrins or an antigen-binding fragment thereof (e.g., a bispecific antibody or an antigen-binding fragment thereof), or a therapeutically effective amount of a pharmaceutical composition of the present invention, thereby treating the epithelial cell carcinoma.

[0022] Another aspect of the present invention relates to a method for treating cancer in a subject requiring cancer treatment, comprising the following steps:

[0023] a) a step of selecting a target having cancer cells rich in integrin αvβ3 and / or integrin α5β1, and selectively immune effector cells (e.g., macrophages); and

[0024] b) a step of treating cancer by administering to a subject an antibody targeting one or more fipronectin-binding integrins or a binding fragment thereof (e.g., a bispecific antibody or an antigen-binding fragment thereof), or a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0025] Another aspect of the present invention relates to a method for treating a fibrotic disease in a subject who requires treatment for a fibrotic disease expressing integrin αvβ3 and / or integrin α5β1, comprising the step of treating the fibrotic disease by administering to the subject an antibody targeting one or more fibronectin-binding integrins or an antigen-binding fragment thereof (e.g., a bispecific antibody or an antigen-binding fragment thereof), or a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0026] Another aspect of the present invention relates to a method for inhibiting collagen organization in a subject, comprising the step of administering a composition targeting fibronectin.

[0027] Another aspect of the present invention relates to a method for disrupting collagen organization already formed in a subject, comprising the step of administering a composition targeting fibronectin.

[0028] Another aspect of the present invention relates to the delivery of a nucleic acid (e.g., siRNA) for inhibiting the expression of a specific integrin receptor subunit, which may be in the form of a nucleic acid, a gene vector construct, or a recombinant viral vector. Inhibition of integrin receptor expression may be any single integrin receptor or a combination comprising integrin αvβ3 and / or integrin α5β1.

[0029] Another aspect of the present invention relates to a method for inhibiting Yes-associated protein 1 (YAP) or a YAP target gene in a subject, comprising the step of administering one or more fibronectin-binding integrins (e.g., the bispecific antibody of the present invention or its antigen-binding fragment, a polynucleotide encoding the antibody or antigen-binding fragment, a vector comprising the polynucleotide, or its antigen-binding fragment) to a subject.

[0030] Another aspect of the present invention relates to the manufacture of a pharmaceutical product for use in the treatment of cancer expressing integrin αvβ3 and integrin α5β1 in a subject, comprising a bispecific antibody or an antigen-binding fragment thereof comprising a first variable domain specifically binding to integrin αvβ3 and a second variable domain specifically binding to integrin α5β1.

[0031] Another aspect of the present invention relates to the use of a pharmaceutical product for the treatment of epithelial cell carcinoma in a subject, comprising a bispecific antibody or an antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin αvβ3 and a second variable domain that specifically binds to integrin α5β1.

[0032] Another aspect of the present invention relates to the manufacture of a pharmaceutical product for the treatment of fibrotic diseases in which a subject expresses integrin αvβ3 and / or integrin α5β1, comprising a bispecific antibody or an antigen-binding fragment thereof comprising a first variable domain specifically binding to integrin αvβ3 and a second variable domain specifically binding to integrin α5β1.

[0033] Another aspect of the present invention provides a use for manufacturing a pharmaceutical product for use in inhibiting the binding of fibronectin to cells in a target, inhibiting collagen organization, reversing collagen organization, and / or inhibiting YAP or YAP target genes, wherein the antibody or its antigen-binding fragment comprises a first variable domain specifically binding to integrin αvβ3 and a second variable domain specifically binding to integrin α5β1.

[0034] Another aspect of the present invention relates to a bispecific antibody or an antigen-binding fragment thereof comprising a first variable domain specifically binding to integrin αvβ3 and a second variable domain specifically binding to integrin α5β1, for use in the treatment of cancer expressing integrin αvβ3 and integrin α5β1 in a subject.

[0035] Another aspect of the present invention provides a bispecific antibody or an antigen-binding fragment thereof comprising a first variable domain specifically binding to integrin αvβ3 and a second variable domain specifically binding to integrin α5β1, for use in the treatment of epithelial cell carcinoma in a subject.

[0036] Another aspect of the present invention provides a bispecific antibody or an antigen-binding fragment thereof comprising a first variable domain specifically binding to integrin αvβ3 and a second variable domain specifically binding to integrin α5β1, for use in the treatment of fibrotic diseases in which a subject expresses integrin αvβ3 and / or integrin α5β1.

[0037] Another aspect of the present invention provides a bispecific antibody or an antigen-binding fragment thereof comprising a first variable domain specifically binding to integrin αvβ3 and a second variable domain specifically binding to integrin α5β1, for use in inhibiting binding to fibronectin in cells in a subject, inhibiting collagen organization, reversing collagen organization, and / or inhibiting YAP or YAP target genes.

[0038] These aspects and other aspects of the invention are presented in more detail in the following detailed description of the invention. Brief explanation of the drawing

[0039] FIGS. 1A to 1B Fibroblasts require integrins α5 and β3 for the formation of fibronectin and collagen I matrix. Fig. 1A This shows images of immunostaining for fibronectin and collagen I in CAF1299 cancer-associated fibroblasts treated with siRNA for 72 hours. The collagen I staining image in the right panel is an enlarged view of the indicated box area. Fig. 1B This shows Western blot results indicating the effects of different siRNA or integrin antibodies on the expression levels of FN, collagen I, α5, and β3 proteins in CAF1299. FIGS. 2A to 2C. Antibodies targeting integrins α5β1 and αvβ3 significantly inhibit the formation of fibronectin and collagen I substrates (confirmed in permeated fibroblasts). Fig. 2A.A bispecific antibody (ABT601) that recognizes αvβ3 and α5β1 heterodimers. The schematic diagram shows the design of a novel bispecific antibody that recognizes two antigens, integrin αvβ3 and integrin α5β1, as monovalents. The Fab domain of the bispecific antibody is identical to the Fab domain of a "control" bivalent (i.e., monospecific) monoclonal antibody that recognizes αvβ3 and α5β1. Fig. 2B. This shows images taken using a confocal microscope after immunostaining CAF1299 cells treated with different antibodies (10 μg / mL) for 72 hours for fibronectin and collagen I. The collagen I immunostaining image in the right panel is an enlarged view of the indicated box area. Fig. 2C. Western blot results showing the effects of different integrin antibodies on the expression levels of FN, collagen I, and GAPDH proteins in CAF1299. Fig. 3. Antibodies targeting integrins α5β1 and αvβ3 significantly inhibit the formation of fibronectin and collagen matrix (confirmed in non-permeable treated fibroblasts). CAF1299 cancer-associated fibroblasts were treated with different antibodies (10 μg / mL) for 72 hours, fixed with 4% PFA, blocked with 1× HBSS buffer containing 5% goat serum, and then immunofluorescence staining for collagen I and fibronectin was performed and images were acquired using a confocal microscope. Fig. 4. Antibodies targeting integrins α5β1 and αvβ3 do not reduce the mRNA expression of FN1 and COL1A1. Quantitative RT-PCR shows the effects of FN siRNA or different integrin antibodies on the mRNA expression levels of FN1 and COL1A1 in cancer-associated fibroblast cell lines CAF1299 and CAF1424. FIGS. 5A to 5B.Targeting integrins α5 and β3 using siRNA or antibodies increases fibroblast secretion of soluble collagen I that is not incorporated into the extracellular matrix. Fig. 5A. Western blot shows the effects of different siRNA or integrin antibodies on the expression levels of FN, collagen I, α5, and β proteins in the cancer-associated fibroblast cell line CAF1299. Fig. 5B. Western blot shows the effect of FN siRNA or integrin antibodies on the expression level of soluble collagen I protein secreted into the supernatant from CAF1299. Fig. 6. Antibodies targeting α5β1 and αvβ3 can disrupt fibronectin and collagen I substrates already formed by fibroblasts. Cancer-associated fibroblasts (CAF1299) were cultured for 72 hours to form fibronectin and collagen I substrates. Subsequently, cells were treated with different integrin antibodies (10 μg / mL) for up to 96 hours (fresh antibodies were added every 48 hours), then fixed with 4% PFA, blocked with 1× HBSS buffer containing 5% goat serum, and then immunofluorescence staining for fibronectin and collagen I was performed and images were acquired using a confocal microscope. Fig. 7.Antibodies targeting α5β1 and αvβ3 can inhibit the production of collagen fibers by fibroblasts. Cancer-associated fibroblasts, CAF1299, were cultured for 72 hours to form fibronectin and collagen I substrates. Subsequently, cells were treated with different antibodies (IgG4, anti-αvβ3 (ABT101), anti-α5β1 (ABT701), or anti-αvβ3 / α5β1 (ABT601); all antibody concentrations were 10 μg / mL) for up to 96 hours (fresh antibodies were added every 48 hours), followed by Picrosirius Red staining to detect fibrillar collagen. Representative brightfield images (×8) acquired from three randomly selected fields of view for each treatment group are shown. Fig. 8. Antibodies targeting α5β1 and αvβ3 can disrupt fibronectin and collagen I substrates already formed by fibroblasts. CAF1299 cancer-associated fibroblasts were cultured for 72 hours to form fibronectin and collagen I substrates. After removing the cells, the cell-free extracellular matrix (ECM) was treated with different integrin antibodies (10 μg / mL) for up to 96 hours (fresh antibodies were added every 48 hours), then fixed with 4% PFA, blocked with 1× HBSS buffer containing 5% goat serum, and then immunofluorescence staining for fibronectin and collagen I was performed. Fig. 9.Antibodies targeting α5β1 and / or αvβ3 do not affect the attachment of fibroblasts to fibronectin or collagen I substrates. CAF1299 cancer-associated fibroblasts were mixed with different integrin antibodies (10 μg / mL each) and seeded into untreated 96-well cell culture plates coated with plasma fibronectin or collagen I. Cells were allowed to adhere to the plates for up to 24 hours, and after staining the attached cells with crystal violet, the absorbance at 560 nm was measured using a plate reader. Fig. 10. Bispecific antibodies exhibit enhanced binding ability to the cell surface of pancreatic tumor cells compared to their respective monospecific antibodies. Human pancreatic ductal adenocarcinoma cells KP4 and PANC1 were cultured with IgG4, anti-αvβ3 (ABT101), anti-αvβ3 / α5β1 (ABT601), or anti-α5β1 (ABT701) (10 μg / mL each), and then cells were fixed at the final stage to perform Fluorescence Activated Cytometry (FACS). The left panel shows the central fluorescence intensity (MFI), and the right panel shows the histograms of each cell treatment group. Fig. 11. In tumor cells cultured on an ECM from which cells formed by cancer-associated fibroblasts cultured in the presence of antibodies targeting α5β1 and αvβ3 have been removed, the expression of Yes-associated protein 1 (YAP) targets is not increased. Western blots show the expression levels of Yes-associated protein 1 (YAP) and two YAP target proteins (CTGF and CYR61) in human pancreatic cancer cells, KP4 or PANC1, cultured on an ECM from which cells formed by CAF cells treated with different integrin antibodies (10 μg / mL) for 24 hours have been removed. All cells were cultured in serum-free medium. Fig. 12. Human lung tumor biopsy tissue in vitro ( ex vivoTreatment with a bispecific antibody reduces collagen content. The collagen area ratio (%) on the 4th day after treating lung tumor tissue sections with ABT701 or ABT601 antibodies was shown and analyzed using QuPath. FIGS. 13A to 13C. Tumor cells overcome isolation stress by utilizing fibronectin produced by CAF. Fig. 13A. CAF promotes tumor initiation via fibronectin (precisional pancreatic cancer model). Luciferase-expressing KP4 PDAC cells were precisionally injected into the pancreas of nude mice, either alone or in combination with an equal number of CAF-1299 cancer-associated fibroblasts. After 3 weeks, luciferase imaging was performed to confirm tumor formation in the pancreas. The table shows the tumor formation rates in 5 mice per group. Fig. 13B. Inhibition of fibronectin expression inhibits the formation of collagen fibers by CAF. CAF-1299 and CAF-1424 cells were treated with scrambled controls or siRNA targeting FN. After 72 hours, the content of FN and COL was determined by immunofluorescence staining. Images represent at least three independent experiments. Fig. 13C. In CAF, inhibition of fibronectin expression increases the amount of soluble collagen secreted into the medium. After culturing CAF-1299 cells for 72 hours, inhibition of fibronectin expression was confirmed by collecting the conditioned medium and cell lysates and performing an immunoblot, and the level of soluble collagen secreted by the cells into the medium was evaluated. Fig. 14.Fibronectin-binding integrins mediate the formation of the ECM. Inhibition of FN or FN-binding integrin expression in CAFs inhibits the formation of FN and COL fibers. CAF-1299 cells were treated with scrambled controls, FN, ITGA5, or siRNAs targeting ITGB3. After 72 hours, FN and COL content was determined by immunofluorescence staining. Images represent at least three independent experiments. Western blots confirm that expression inhibition occurred. FIGS. 15A to 15B. ABT601 enhances the ability to target fibronectin-binding integrins on CAFs. Fig. 15A. The cell binding ability of ABT601 was compared with commercial antibodies and control antibodies. Flow cytometry results indicate the binding of each antibody to CAF-1299 and CAF-1424 cells. Fig. 15B. ABT601 exhibits enhanced binding ability to CAF compared to its constituent mAbs. The graph shows the median fluorescence intensity (MFI) for antibody binding to CAF-1299 and CAF-1424 cells. Error bars represent the standard deviations obtained from three independent experiments. FIGS. 16A to 16C. Double blockade of integrins αvβ3 / α5β1 can prevent and reverse the formation of extracellular matrix (ECM) by CAF. Fig. 16A. Function-blocking antibodies targeting the FN receptor inhibit the formation of FN / COL fibers induced by CAF. After incubating CAF-1299 cells with control IgG or the labeled antibodies for 72 hours, immunostaining was performed to confirm FN and COL. Images represent at least three independent experiments. The graphs show the mean ± standard deviation of the values ​​normalized to the IgG control after measuring the percentage staining area (% area) of each marker in each experiment. * indicates P < 0.05 in the analysis using a one-sample t-test. Fig. 16B.Antibodies targeting integrins can degrade the ECM previously formed by CAF. CAF-1299 cells were cultured to form an ECM for 72 hours, after which they were cultured for an additional 72 hours with the addition of control IgG or the labeled antibodies. Subsequently, immunostaining was performed to confirm FN and COL. The graph shows the mean ± standard deviation of the values ​​normalized to the IgG control group after measuring the percentage of stained area (% area) for each marker in each experiment. * indicates P < 0.05 in the analysis using the one-sample t-test. Fig. 16C. ABT601 inhibits the activity of YAP, a mechanoreceptor that promotes stem cell characteristics. CAF-1299 cells were cultured to form an ECM for 72 hours, after which antibodies were added and treated for another 72 hours. Subsequently, the cells were removed to leave only the cell-free ECM, upon which PANC1 cells were cultured. After 24 hours, PANC1 cells were lysed, and an immunoblot was performed to detect the expression of CTGF, a YAP target protein. The blot represents at least three independent experiments. FIGS. 17A to 17C. Inhibiting FN-binding integrin suppresses the tumor initiation-promoting ability of CAF. Fig. 17A. CAF promotes tumor initiation through FN and FN-binding integrins (subcutaneous graft tumor model). PDAC cells, specifically PANC1, were injected subcutaneously into the flanks of nude mice, either alone or in combination with CAF-1299. Mice were observed twice a week to determine the time at which provable tumors first developed. The graph shows the tumor formation rate over time in 10 to 12 mice per group, with the tumor formation threshold set at 100 mm³ (calculated as length × width²). Tumors were excised at the end of the experiment (day 56) and prepared for histological analysis. Fig. 17B. Treatment with bispecific antibodies reduces tumor stroma and increases necrosis. Tumor tissue sections were stained with H&E. Stroma (S ) and necrosis( N The ) area was marked. Fig. 17C. Tumor hardness is reduced by bispecific antibody treatment. Tissue hardness was evaluated by analyzing frozen tumor sections using an atomic force microscope (AFM). Each point represents the average value for each 20 μm × 20 μm region of interest (ROI). Two tumors were analyzed in each group, and 15 ROIs were evaluated for each tumor. FIGS. 18A to 18D. The fibrotic effect of human CAF injected with tumor cells is reduced by ABT601 treatment. FIGS. 18A to 18D. Tumors treated with ABT601 exhibit a state of reduced fibronectin, fibrosis, and angiogenesis generated by CAF. Using serial formalin-fixed paraffin-embedded (FFPE) sections of the tumor, human-specific fibronectin (human-specific FN, hFN) ( Fig. 18A ) or CD31( Fig. 18D ) was detected by immunohistochemical staining, and the arrows indicate the corresponding signals. Trichrome ( Fig. 18B ) and Picrosirius Red( Fig. 18C Collagen was visualized using ). The graph shows the results of quantitative analysis of IHC using QuPath, and each point represents the mean value for each analyzed tumor section. The P-value was calculated using Student's t-test. Fig. 19. Fibronectin generated by CAFs promotes tumor initiation (summary schematic). Cancer-associated fibroblasts produce a dense and reactive matrix that supports tumor initiation and progression. The results of this specification indicate that the association between cell surface integrins and FNs plays a key role in the formation of tumor-promoting ECM, thereby leading to various in vitro ( in vitro ) and in vivo( in vivoThis study demonstrates that CAF can explain the ability to support tumor initiation in a model. Inhibiting the expression of integrins αvβ3 and / or α5β1, or blocking them with antibodies, can inhibit the formation of FN fibers. FN fibers are known to function as scaffolds that mediate the formation of other ECM proteins and the incorporation of tumor-promoting secreted factors. Inhibiting the expression of integrins αvβ3 / α5β1 or dual-blocking them using a novel bispecific antibody (ABT601) can block this chain of processes by inhibiting the initial formation of FN fibers. This approach can be widely utilized as a novel strategy targeting abnormal fibrosis that exacerbates the progression of cancer and fibrotic diseases. FIGS. 20A to 20D. ABT601 reduces the fibrotic response in a metabolic dysfunction-associated steatohepatitis (MASH) model. Fig. 20A Primary hepatocytes isolated from human donors were mixed with ABT601 and cultured for 7 days to form spheroids, followed by stimulation with a MASH cocktail. On the 14th day, spheroids and supernatant were collected and analyzed. Fig. 20B On day 14, brightfield images were acquired, and the diameter of the spheroid was measured and normalized using ImageJ software. Fig. 20C ) An ELISA was performed to measure the protein concentration of Collagen I in the spheroid supernatant. Fig. 20D qPCR was performed on spheroid lysates to analyze the expression of genes important for the fibrosis response. Specific details for implementing the invention

[0040] The present invention is described in more detail below. However, this description is not intended to enumerate in detail all different ways in which the present invention may be implemented or all features that may be added to the present invention. For example, features illustrated in relation to one embodiment may be included in another embodiment, and features illustrated in relation to a specific embodiment may be omitted from that embodiment. Furthermore, in light of the disclosure of this specification, numerous modifications and additions to the various embodiments presented herein will be obvious to those skilled in the art without departing from the scope of the present invention. Accordingly, the following detailed description is intended to illustrate some specific embodiments of the present invention and is not intended to describe all possible arrangements, combinations, and variations of the present invention.

[0041] Unless otherwise indicated in the context, the various features of the invention described herein are specifically intended to be used in any combination. Additionally, the invention includes the possibility that any feature or combination of features described herein may be excluded or omitted in some embodiments. For example, where the specification describes a composite as comprising components A, B, and C, it is specifically intended that any one of A, B, or C, or any combination thereof, may be omitted or excluded from the scope of rights, either alone or in any combination.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Furthermore, the terms used herein are intended only to describe specific embodiments and are not intended to limit the present invention.

[0043] Unless otherwise specified, standard methods known to those skilled in the art may be used for the preparation of recombinant or synthetic polypeptides, antibodies or their antigen-binding fragments, the manipulation of nucleic acid sequences, and the preparation of transformed cells. These techniques are well known to those skilled in the art. For example, Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual , 4th Ed. (Cold Spring Harbor, NY); Ausubel et al. Current Protocols in Molecular Biology You may refer to (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).

[0044] All publications, patent applications, patents, nucleotide sequences, amino acid sequences, and other references mentioned in this specification are incorporated herein by reference in their entirety.

[0045] Unless the context clearly indicates otherwise, the singular expressions "a," "an," and "the" used in this specification and the appended claims are intended to include the plural forms.

[0046] As used herein, "and / or" means all possible combinations of one or more of the related items, including such combinations, and in the case where interpreted in an optional sense ("or"), includes cases where such combinations are absent.

[0047] Additionally, the present invention includes the fact that in some embodiments, any feature or combination of features described herein may be excluded or omitted.

[0048] Additionally, when the term "drug" as used herein refers to a measurable value such as the amount, dosage, time, temperature, etc. of the compound or formulation of the present invention, it means that the value includes a variation range of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% with respect to the specified value.

[0049] Unless otherwise specified, all numerical values ​​representing characteristics such as the amount of ingredients and reaction conditions used in the specification and claims should be understood as being modified by the term "about" in all cases. Accordingly, unless otherwise specified, numerical parameters described in this specification and claims are approximations that may vary depending on the desired characteristics to be achieved by the subject matter of this disclosure.

[0050] The ranges used in this specification may be expressed as a range from one specific value to another specific value. Additionally, this specification discloses multiple numerical values, and each numerical value should be understood as disclosing not only the value itself but also a value to which "approximately" is added. For example, if a value of "10" is disclosed, it should be understood that "approximately 10" is also disclosed. Additionally, each numerical value between two disclosed specific values ​​should also be understood as disclosed. For example, if 10 and 15 are disclosed, it should be understood that 11, 12, 13, and 14 are also disclosed.

[0051] The transitional phrase “essentially composed” means that the scope of the claim should be interpreted as including specific substances or steps described in the claim, and also including substances or steps that do not substantially affect the fundamental and novel characteristics of the claimed invention.

[0052] In the present invention, the term “essentially composed” (and its grammatical variations) applied to a polynucleotide or polypeptide sequence means a polynucleotide or polypeptide composed of a described sequence (e.g., SEQ ID NO) and a total of 10 or fewer additional nucleotides or amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the 5’ end and / or 3’ end of the described sequence, or at the N-end and / or C-end, or between the two ends (e.g., between domains), such that the function of the polynucleotide or polypeptide is not substantially altered. Here, a total of 10 or fewer additional nucleotides or amino acids means the sum of the numbers of all added nucleotides or amino acids.

[0053] As used in this specification, the term “polypeptide” includes both peptides and proteins unless otherwise specified.

[0054] The term “chimeric” refers to a molecule that has two or more parts that are not naturally found together within a single molecule.

[0055] "Nucleic acid" or "nucleotide sequence" means a sequence of nucleotide bases, and may be RNA, DNA, or DNA-RNA hybrid sequences (including both naturally occurring nucleotides and non-naturally occurring nucleotides), but preferably a single-stranded or double-stranded DNA sequence.

[0056] As used herein, the term “separated” means that a molecule, such as, for example, a protein, polynucleotide, or cell, is separated from or substantially free from other components of at least part of a naturally occurring organism or virus. Examples of other components include structural components of the cell or other polypeptides or nucleic acids that are typically present together with the molecule. Additionally, the term includes synthetically produced molecules.

[0057] In this specification, "treat," "treating," or "treatment of" (or grammatically equivalent terms) means that the severity of the subject's condition is reduced or at least partially improved or alleviated, and / or one or more clinical symptoms are at least partially relieved, alleviated, or reduced, and / or the progression of said condition is delayed.

[0058] As used herein, the terms “prevent,” “prevents,” or “prevention” and “inhibit,” “inhibits,” or “inhibition” and their grammatical equivalents) do not mean that the disease is completely eliminated, but include all forms of preventive treatment that reduce the incidence of the disease, delay the onset of the disease, and / or reduce symptoms associated with the disease after onset. Inhibition may include a reduction in the incidence of the condition (e.g., tumor growth or fibrosis) by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. Delay in onset of the condition may include a delay in onset of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or more.

[0059] As used herein, "effective amount," "preventively effective amount," or "therapeutically effective amount" means an amount sufficient to provide any improvement or benefit to the subject. In other words, "effective amount," "preventively effective amount," or "therapeutically effective amount" means an amount that provides a certain degree of delay, alleviation, relief, or reduction of at least one clinical symptom in the subject. Those skilled in the art will understand that the effect does not need to reach a complete or therapeutic level, and is sufficient as long as some benefit is provided to the subject.

[0060] As used herein, the term “composition targeting fibronectin” means a composition capable of inhibiting the binding of fibronectin to one or more corresponding receptors on a cell surface. A composition targeting fibronectin may directly block or interfere with the binding interaction between fibronectin and one or more corresponding receptors, or may inhibit the expression of fibronectin or one or more corresponding receptors. In some embodiments, one or more corresponding receptors of fibronectin are selected from the group consisting of αvβ3, αvβ6, and α5β1.

[0061] The terms “specifically bind” or “specifically binding” as used herein mean that, with respect to the bispecific antibody of the present invention or its antigen-binding fragment, the agent binds to the epitope of the target (including one or more epitopes) but does not substantially bind to other unrelated epitopes or molecules. In some embodiments, the term refers to an agent that exhibits at least about 60%, e.g., at least about 70%, 80%, 90%, or 95% binding to a target epitope (e.g., αvβ3, αvβ6, or α5β1) compared to binding to other unrelated epitopes or molecules. The term “specifically binding” does not necessarily mean that the agent binds exclusively to the intended target. Rather, the agent is considered to bind specifically if its affinity for the intended target is at least about 2 times higher than its affinity for unrelated epitopes or molecules. Preferably, the affinity of the agent for the target molecule is at least about 5 times higher than its affinity for the non-target molecule, e.g., at least about 10 times, at least about 25 times, at least about 50 times, or at least about 100 times higher. In some embodiments, the specific between the bispecific antibody or its antigen-binding fragment and the antigen The combination is at least about 10 6 M -1 , for example, At least about 10 7 M -1 , or at least about 10 8 M -1 to about 10 9 M -1 or 10 10 M -1 It refers to the bonding affinity of.

[0062] The terms used in this specification, “gene interference” or “gene expression interference,” mean delivering nucleic acids into a cell solely for the purpose of inhibiting gene expression through interaction with mRNA.

[0063] As used herein, the term "isolation stress" refers to the microenvironmental stress experienced by a single tumor cell that is not surrounded by other tumor cells and is located in a surrounding environment including normal cells and the extracellular matrix, in contrast to areas with highly reactive tumor matrix or immune cell / vascular infiltration typically observed in established tumors. Examples of tumor cells experiencing isolation stress include tumor initiation cells at primary and metastatic sites, dormant tumor cells within normal tissues, circulating tumor cells within the blood / lymphatic vessels, and single tumor cells in migration or infiltration. Such microenvironmental stress includes, but is not limited to, loss of intercellular contact, isolation from appropriate matrix, hypoxia, and nutrient deprivation.

[0064] bispecific antibody or its antigen-binding fragment

[0065] The first aspect of the present invention relates to a bispecific antibody or an antigen-binding fragment thereof comprising two different variable domains that bind to two different integrins selected from α5β1, αvβ3, and αvβ6. In some embodiments, the present invention relates to a bispecific antibody or an antigen-binding fragment thereof comprising a first variable domain that specifically binds to integrin αvβ3 or integrin αvβ6 and a second variable domain that specifically binds to integrin α5β1 or integrin αvβ6, wherein the first variable domain and the second variable domain are different from each other, i.e., bind to two different integrins. In some embodiments, the bispecific antibody or an antigen-binding fragment thereof comprises a first variable domain that specifically binds to integrin αvβ3 and a second variable domain that specifically binds to integrin α5β1. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises a first variable domain that specifically binds to integrin αvβ6 and a second variable domain that specifically binds to integrin α5β1. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises a first variable domain that specifically binds to integrin αvβ3 and a second variable domain that specifically binds to integrin αvβ6.

[0066] A bispecific antibody or its antigen-binding fragment may be any structure capable of binding to two antigens on the same cell or different cells. In some embodiments, the bispecific antibody or its antigen-binding fragment exhibits enhanced binding ability to cells containing two antigens compared to the constituent antibodies used in the preparation of the bispecific antibody or its antigen-binding fragment. In some embodiments, the variable domain of the bispecific antibody or its antigen-binding fragment binds to cells containing two antigens with a synergistic effect. As used herein, "synergistically," "synergistic effect," or any grammatical variation thereof means that the binding and / or biological effect of the bispecific antibody or its antigen-binding fragment exceeds the simple sum of the binding and / or biological effects of the antibodies from which the variable domain of the bispecific antibody or its antigen-binding fragment is derived. In some embodiments, one or more portions of the bispecific antibody or its antigen-binding fragment are composed of antibody fragments.

[0067] In a specific embodiment, the first domain of the bispecific antibody or its antigen-binding fragment is the first antibody variable domain. In a specific embodiment, the second domain of the bispecific antibody or its antigen-binding fragment is the second antibody variable domain. In some embodiments, both domains are antibody domains. In some embodiments, the first domain is a humanized antibody domain or a human antibody domain. In some embodiments, the second domain is a humanized antibody domain or a human antibody domain. In some embodiments, the first domain and the second domain are a humanized antibody domain or a human antibody domain. Examples of methods for producing bispecific antibodies are described, for example, at scientistlive.com / content / bispecific-antibodies. In one embodiment, a CrossMab approach may be used to produce a bispecific antibody, which may involve crossing antibody domains within one arm of a bispecific IgG antibody, for example, a constant CH1-CL domain or a variable heavy chain (VH)-variable light chain (VL) domain within a Fab domain on one arm of a bispecific IgG antibody. The above-mentioned bispecific antibody may include additional engineering strategies, including Knob-in-Hole (KiH) engineering of the Fc domain as described herein.

[0068] In some embodiments, the bispecific antibody or its antigen-binding fragment further comprises the Fc domain of an IgG antibody. In one embodiment, the Fc domain is the Fc domain of an IgG1 or IgG4 antibody. In one embodiment, the Fc domain is engineered to substitute a small amino acid with a large amino acid in the CH3 domain of one chain and a large amino acid with a small amino acid in the opposite chain to construct a KiH Fc domain. In some embodiments, the amino acid sequence of the bispecific antibody or its antigen-binding fragment comprises one or more of the T363S, V366A, and Y404V (based on the Eu numbering system) mutations. In some embodiments, the amino acid sequence of the bispecific antibody or its antigen-binding fragment comprises the T366W (based on the Eu numbering system) mutation in the Fc domain of the second heavy chain of the bispecific antibody.

[0069] In some embodiments, the first domain and the second domain specifically bind to integrins αvβ3, αvβ6, and / or α5β1 present on the surface of a cell, e.g., a cancer cell, e.g., an epithelial cancer cell. In some embodiments, the first domain and the second domain specifically bind to integrins αvβ3 and α5β1 present on the surface of a cell, e.g., a cancer cell, e.g., an epithelial cancer cell. In some embodiments, the first domain and the second domain specifically bind to integrins αvβ6 and α5β1 present on the surface of a cell, e.g., a cancer cell, e.g., an epithelial cancer cell. In some embodiments, the first domain and the second domain specifically bind to integrins αvβ3 and αvβ6 present on the surface of a cell, e.g., a cancer cell, e.g., an epithelial cancer cell. In some embodiments, the antigen is a receptor found on the surface of a mesenchymal-like tumor cell. In some embodiments, the first variable domain specifically binds to integrin αvβ3. In some embodiments, the second variable domain specifically binds to integrin α5β1.

[0070] In some embodiments, the first domain and / or the second domain binds to an antigen that is not present, is present at a low level, or is present at a lower level on the surface of normal epithelial cells compared to epithelial cancer cells. In some embodiments, the antigen may be an antigen that is not present, is present at a low level, or is present at a lower level on the surface of epithelial cancer cells compared to cells at which epithelial cancer cells begin to metastasize to mesenchymal cells. In some embodiments, the antigen may be an antigen that is present only after epithelial cancer cells begin to metastasize to mesenchymal cells, or is present at an increased level. In some embodiments, the antigen is a mesenchymal cell antigen that is not present or is present only at a low level on epithelial cancer cells until epithelial cancer cells begin to metastasize to mesenchymal cells.

[0071] In certain embodiments, the first domain comprises the Fab domain of an antibody, is substantially composed of the Fab domain of an antibody, or is composed of the Fab domain of an antibody. The Fab domain may be derived from an antibody of any antibody isotype. In some embodiments, the first domain comprises the Fab domain of an IgG antibody, e.g., an IgG1 or IgG4 antibody. In some embodiments, the first domain comprises the amino acid sequence of etaracizumab. In some embodiments, the first domain comprises an amino acid sequence comprising the VH domain and CL of the light chain of etaracizumab, and a heavy chain comprising the VL of the light chain and CH1 of the heavy chain. In some embodiments, the first domain Sequence No. 1 A heavy chain VH-(AS)-hk.LC.h4.HC.S228P.T366W comprising the amino acid sequence of or a sequence at least 90% identical thereto, e.g., a sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value in between), and Sequence No. 2It includes a light chain VL-(SS)-h1v2.CH1 comprising an amino acid sequence or a sequence that is at least 90% identical thereto, for example, a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value in between).

[0072] In certain embodiments, the second domain comprises, substantially comprises, or consists of the Fab domain of the antibody. The Fab domain may be derived from any antibody isoform. In some embodiments, the second domain comprises the Fab domain of an IgG antibody, e.g., an IgG1 or IgG4 antibody. In some embodiments, the second domain comprises the amino acid sequence of volociximab. In some embodiments, the second domain comprises an amino acid sequence comprising the VH domain and CH1 of the light chain of volociximab and a heavy chain comprising the VL of the light chain and the CL of the light chain, and optionally may comprise one or more mutations in the Fc domain of the heavy chain. In some embodiments, the second domain Sequence No. 3 A heavy chain VH-h4.HC.S228P.T363S.V366A.Y404V comprising the amino acid sequence of or a sequence at least 90% identical thereto, e.g., a sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value in between), and Sequence No. 4 It includes a light chain VL-hk.LC comprising an amino acid sequence or a sequence that is at least 90% identical thereto, for example, a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value in between).

[0073] In certain embodiments, the antibody is a chimeric monoclonal antibody that specifically binds to integrin α5β1 and inhibits its functional activity. In some embodiments, the antibody is Sequence No. 5 A heavy chain VH-h4.HC.S228P comprising the amino acid sequence of or a sequence at least 90% identical thereto, e.g., a sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value in between), and Sequence number 6 It includes a light chain VL-hk.LC comprising an amino acid sequence or a sequence that is at least 90% identical thereto, for example, a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value in between).

[0074] A bispecific antibody or its antigen-binding fragment may include sequence modifications known to enhance the properties of the antibody, e.g., stability, or alter the antibody's binding to the Fc-gamma receptor. In some embodiments, said sequence modifications provide selective cell recruitment, e.g., NK cell recruitment, macrophage recruitment, or non-recruitment of effector cells. Thus, the properties of the therapeutic antibody can be modified to enhance or inhibit binding to specific types of immune effector cells using increasingly diverse glycoengineering and Fc engineering approaches (Saxena & Wu (2016) Front. Immunol. 7:580). In one embodiment, the sequence modification provides a variant in which the Fc portion of the therapeutic antibody promotes binding to Fcγ, the only Fc receptor expressed on NK cells (Lazar et al. (2006) Appl. Biol. Sci. 103(11):4005-4010). In some embodiments, the sequence modification enhances binding to macrophages by including a G236A Fc mutation that promotes binding to Fcγ (Richards et al. (2008) Mol. Cancer Ther. 7(8):2517-27) or a bispecific antibody that recruits macrophages via FcαRI (CD89) (Li et al. (2018) OncoImmunol. 7(1):e1380142).

[0075] In some embodiments, the amino acid sequence of a bispecific antibody or its antigen-binding fragment contains an S228P (based on the Eu numbering system) mutation in the hinge region. In some embodiments, the amino acid sequence contains an S228P mutation in the Fc domain of an IgG (e.g., IgG1, IgG4) antibody, enabling selective macrophage recruitment. In some embodiments, the amino acid sequence contains L234A, L235A, and P329G (LALA-PG) mutations in the Fc domain of an IgG (e.g., IgG1, IgG4) antibody, providing non-recruitment of effector cells. In some embodiments, the amino acid sequence contains mutations selected from the following:

[0076] a) S239D / A330L / I332E;

[0077] b) I332E;

[0078] c) G236A / S239D / I332E;

[0079] d) G236A;

[0080] e) N297A / E382V / M428I;

[0081] f) M252Y / S254T / T256E;

[0082] g) Q295R / L328W / A330V / P331A / I332Y / E382V / M428I;

[0083] h) L234A / L235A / P329G;

[0084] i) M428L / N434S;

[0085] j) L234A / L235A / P331S;

[0086] k) L234A / L235A / P329G / M252Y / S254T / T256E;

[0087] l) S298A / E333A / K334 / A;

[0088] m) S239D / I332E;

[0089] n) G236A / S239D / A330L / I332E;

[0090] o) S239D / I332E / G236A;

[0091] p) L234Y / G236W / S298A;

[0092] q) F243L / R292P / Y300L / V305I / P396L;

[0093] r) K326W / E333S;

[0094] s) K326A / E333A;

[0095] t) K326M / E333S;

[0096] u) C221D / D222C;

[0097] v) S267E / H268F / S324W;

[0098] w) H268F / S324W;

[0099] x) E345R

[0100] y) R435H;

[0101] z) N434A;

[0102] aa) M252Y / S254T / T256E;

[0103] ab) M428L / N434S;

[0104] ac) T252L / T / 253S / T254F;

[0105] ad) E294delta / T307P / N434Y;

[0106] ae) T256N / A378V / S383N / N434Y;

[0107] af) E294delta

[0108] ag) L235E;

[0109] ah) L234A / L235A;

[0110] ai) S228P / L235E;

[0111] aj) P331S / L234E / L225F;

[0112] ak) D265A;

[0113] al) G237A;

[0114] am) E318A;

[0115] an) E233P;

[0116] ao) G236R / L328R;

[0117] ap) H268Q / V309L / A330S / P331S;

[0118] aq) L234A / L235A / G237A / P238S / H268A / A330S / P331S;

[0119] ar) A330L;

[0120] as) D270A;

[0121] at) K322A;

[0122] au) P329A;

[0123] av) P331A;

[0124] aw V264A;

[0125] ax) F241A;

[0126] ay) N297A or G or N

[0127] az) S228P / F234A / L235A; or

[0128] ba) Any combination of the above a) to az);

[0129] (Based on the Eu numbering system) and may or may not include the S228P mutation.

[0130] The following description is intended to provide a general overview of the techniques available for the production of antibodies. However, those skilled in the art will recognize that various variations of the method described below are known.

[0131] As used in the present invention, the terms “antibody” or “antibodies” refer to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Antibodies may be monoclonal, oligoclonal, or polyclonal, may have origins of any species including (e.g.) mouse, rat, hamster, rabbit, horse, cattle, goat, sheep, pig, camel, monkey, or human, and may be chimeric antibodies or humanized antibodies. For example, Walker et al. (1989) Mol You may refer to Immunol. 26:403. The antibody may be a recombinant monoclonal antibody prepared according to the method disclosed in U.S. Patent No. 4,474,893 or No. 4,816,567. Additionally, the antibody may be chemically synthesized according to the method disclosed in U.S. Patent No. 4,676,980.

[0132] Antibody fragments included within the scope of the present invention include, for example, Fab, Fab′, F(ab)2, and Fv fragments; domain antibodies, diabodies; vaccibodies, linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. These fragments may be prepared by known techniques. For example, the F(ab')2 fragment may be produced by digesting antibody molecules with pepsin, and the Fab fragment may be produced by reducing the disulfide bonds of the F(ab')2 fragment. Alternatively, a Fab expression library may be constructed to rapidly and easily identify monoclonal Fab fragments with desired specificity (Huse et al. (1989) Science 254:1275). In some embodiments, the term “antibody fragment” as used in the present invention may also include any protein construct capable of binding to a target antigen.

[0133] The antibodies of the present invention may be modified or mutated for compatibility with species other than the species in which the antibodies were produced. For example, the antibodies may be humanized or camelized. A humanized form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab′F(ab′)2, or other antigen-binding subsequences of the antibody) comprising a minimal sequence derived from a non-human immunoglobulin. The humanized antibody comprises a human immunoglobulin in which residues of the complementarity determining region (CDR) of the recipient antibody are substituted with residues of the donor antibody (CDR) of a non-human species, such as mouse, rat, or rabbit, having the desired specificity, affinity, and binding ability. In some cases, Fv framework residues of the human immunoglobulin are substituted with corresponding non-human residues. The humanized antibody may also include residues not found in either the recipient antibody or the introduced CDR or framework sequence. Generally, humanizing antibodies comprise at least one, and typically two, nearly all of the variable domains, wherein all or nearly all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or nearly all of the framework (FR) regions (i.e., the sequences between the CDR regions) are human immunoglobulin consensus sequences. Humanizing antibodies may also be superhumanizing antibodies in which only two CDRs are of non-human origin (US Pat. No. 7,087,409). Humanizing antibodies also preferably comprise a certain portion of the constant region (Fc) of the immunoglobulin, typically the constant region of the human immunoglobulin (Jones et al. (1986) Nature 321:522; Riechmann et al. (1988) Nature 332:323; Presta (1992) Curr . Op. Struct. Biol. 2:593).

[0134] Methods for humanizing non-human antibodies are well known in the art. Generally, one or more amino acid residues from non-human sources are introduced into humanized antibodies. These non-human amino acid residues are commonly referred to as "introductory residues" and are typically taken from the "introductory variable domain." Humanization is essentially the method of Winter et al. (Jones et al. (1986) Nature 321:522; Riechmann et al. (1988) Nature This can be done in accordance with Verhoeyen et al. (1988) Science 239:1534) and by substituting the rodent CDR or CDR sequence with the corresponding sequence of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), which are antibodies in which substantially less of the complete human variable domain is replaced with the corresponding sequence of a non-human species. In practice, humanized antibodies are generally obtained by substituting some CDR residues (e.g., all or part of the CDR) and, in some cases, some skeletal residues from a human antibody with residues from a similar site of a rodent antibody.

[0135] Human antibodies are phage display libraries (Hoogenboom & Winter (1991) J. Mol. Biol . 227:381; Marks et al. (1991) J. Mol. Biol It can also be produced using various known techniques, including . 222:581). The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77 and Boerner et al. (1991) J. Immunol.147:86). Likewise, human antibodies can be produced by introducing a human immunoglobulin locus into transgenic animals, such as mice, in which the endogenous immunoglobulin gene is partially or completely inactivated. Upon immune stimulation, the production of human antibodies is observed that are very similar to those observed in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patents No. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, and in the following academic literature: Marks et al. (1992) Bio / Technology 10:779; Lonberg et al. (1994) Nature 368:856; Morrison (1994) Nature 368:812; Fishwild et al. (1996) Nature Biotechnol. 14:845; Neuberger (1996) Nature Biotechnol. 14:826; Lonberg & Huszar (1995) Intern. Rev. Immunol . 13:65.

[0136] Immunogens (antigens) are used to generate antibodies that specifically react with target polypeptides. For example, recombinant or synthetic polypeptides and peptides having a length of at least 5 amino acids (e.g., at least 7 or 10) or longer are preferred immunogens for generating monoclonal or polyclonal antibodies. In one embodiment, an immunogenic polypeptide conjugate is further included as an immunogen. The peptide is used in a pure, partially pure, or impure form. Polypeptides and epitopes suitable for target pathogens and sperm are well known in the art. Polynucleotide and polypeptide sequences can be identified in public sequence databases such as GENBANK® / GENPEPT®. A number of antibodies that specifically bind to target cancer cell antigens are described in the art, and these can be used as starting materials for producing the antibodies of the present invention. Alternatively, novel antibodies against target antigens can be generated using the techniques described herein and techniques well known in the art.

[0137] The recombinant polypeptide is expressed in eukaryotic or prokaryotic cells and purified using standard techniques. Subsequently, the polypeptide or its synthetic form is injected into an animal capable of producing antibodies. The presence and amount of the polypeptide can be measured by generating monoclonal or polyclonal antibodies and applying them to an immunoassay.

[0138] Methods for producing polyclonal antibodies are well known to those skilled in the art. Briefly, an immunogen, e.g., a purified or synthetic peptide, a peptide conjugated to a suitable carrier (e.g., glutathione-S-transferase, keyholimpet hemocyanin, etc.), or a peptide contained in an immune vector such as a recombinant vaccinia virus is selectively mixed with an adjuvant, and animals are immunized with the mixture. The animal's immune response to the immunogen agent is monitored by collecting blood samples and measuring the reaction titer for the peptide of interest. When the antibody titer for the immunogen appears appropriately high, blood is collected from the animal to prepare an antiserum. If necessary, the antiserum is further fractionated to concentrate the antibodies reacting with the peptide. Antibodies against the polypeptide include a conjugated fragment and a single-stranded recombinant form thereof, and are prepared by immunizing animals using an immunogenic conjugate in which the polypeptide is covalently bonded (conjugated) to a carrier protein, for example, as described above. Generally, the immunogen of interest is a polypeptide having a length of about 10 or more amino acids, in another embodiment the polypeptide has a length of about 20 or more amino acids, and in yet another embodiment the fragment has a length of about 30 or more amino acids. The immunogenic conjugate is generally prepared by conjugating the polypeptide to a carrier protein (e.g., in the form of a fusion protein) or alternatively recombinantly expressed in an immunization vector.

[0139] Monoclonal antibodies are produced from cells that secrete the desired antibody. These antibodies are screened to determine whether they bind to normal or modified peptides, or to determine agonist or antagonist activity. Specific monoclonal and polyclonal antibodies are generally K123 at a concentration of approximately 50 mM or less. D It binds as, for example, K of about 1 mM or less D It binds, for example, K of about 0.1 mM or less. DThey bind to or have a higher binding affinity. In some cases, it is desirable to produce monoclonal antibodies from various mammalian hosts, such as rodents, lagomorphs, primates, and humans. The technology for producing such monoclonal antibodies is described by Kohler & Milstein (1975). Nature It is described in 256:495-497. Briefly summarized, this method proceeds by injecting an immunogen, such as an immunogenic peptide, either alone or selectively linked to a carrier protein, into an animal. The animal is then sacrificed, and cells are collected from the spleen and fused with myeloma cells. As a result, hybrid cells capable of proliferating in vitro, namely "hybridomas," are produced. The hybridoma population is then screened to isolate individual clones that secrete a monoclonal antibody against the immunogen. The individual antibody species obtained in this manner are the product of immortalized and cloned single B cells generated in the immune animal in response to a specific site recognized on the immunogenic substance.

[0140] Alternative methods for immortalization include transformation by Epstein-Barr virus, oncogenes, or retroviruses, or other methods known in the art. Colonies derived from single immortalized cells are selected to determine whether they produce antibodies with desired antigen specificity and affinity, and the yield of monoclonal antibodies produced from these cells is enhanced through various techniques, including injecting them into the peritoneal cavity of a vertebrate (preferably mammalian) host. The polypeptides and antibodies of the present invention may be used with or without modification and include chimeric antibodies such as humanized mouse antibodies. Other suitable techniques include methods for selecting recombinant antibody libraries from phages or similar vectors. Techniques regarding this are described in Huse et al. (1989). Science 246:1275-1281 and Ward et al. (1989) Nature It is listed in 341:544-546.

[0141] Antibodies specific to the target polypeptide can also be obtained by phage display technology known in the art.

[0142] The present invention also provides a polynucleotide encoding the bispecific antibody of the present invention or the antigen-binding fragment thereof.

[0143] Additionally, a vector comprising a polynucleotide of the present invention is provided. The vector comprises, but is not limited to, a plasmid vector, a phage vector, a virus vector, or a cosmid vector.

[0144] In some embodiments, the present invention provides a host cell comprising the polynucleotide and / or vector of the present invention. The host cell may be a eukaryotic or prokaryotic cell and may be used to express the bispecific antibody or its antigen-binding fragment, or for other purposes.

[0145] Another aspect of the present invention relates to a composition comprising a carrier and a bispecific antibody of the present invention or an antigen-binding fragment thereof. In some embodiments, the composition is a pharmaceutical composition, and the carrier is a pharmaceutically acceptable carrier.

[0146] In some embodiments, the pharmaceutical composition may further comprise additional therapeutic agents, e.g., chemotherapy agents. Agents useful for treating cancer include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine, vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) Antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) Enzymes (e.g., L-asparaginase); 5) Biological response modifiers (e.g., interferon-alfa); 6) Platinum coordinating complexes (e.g., cisplatin and carboplatin); 7) Anthracenediones (e.g., mitoxantrone); 8) Substituted ureas (e.g., hydroxyurea); 9) Methylhydrazine derivatives (e.g.,Procarbazine (N-methylhydrazine; MIH); 10) Adrenocortical suppressants (e.g., mitotane (o,p'-DDD) and aminoglutethimide); 11) Adrenocorticosteroids (e.g., prednisone); 12) Progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g., flutamide); and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide). In another embodiment, the formulation of the present invention is administered in combination with anti-angiogenesis agents. These angiogenesis inhibitors include antibodies against VEGF (e.g., bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®)) and other promoters of angiogenesis (e.g., bFGF,Antibodies against angiopoietin-1), alpha-v / beta-3 vascular integrin (e.g., VITAXIN), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alpha conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation formulation)(ABRAXANE®), soy isoflavone (Genistein), tamoxifen citrate, thalidomide, ADH-1 (EXHERIN), AG-013736, AMG-706, AZD2171, sorafenib tosylate, BMS-582664, CHIR-265, pazopanib, PI-88, vatalanib, everolimus, suramin, sunitinib malate, XL184, ZD6474, ATN-161, cilenigtide, celecoxib, or any combination thereof, including but not limited thereto It is not the case. In another embodiment, the formulation of the present invention comprises one or more therapeutic antibodies,For example, it is administered in combination with an anticancer antibody or an antibody against an immune checkpoint. In another embodiment, the formulation of the present invention is administered in combination with one or more immune checkpoint inhibitors. The immune checkpoint inhibitor may be any molecule that inhibits an immune checkpoint. Immune checkpoints are well known in the art and include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA4, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, A2AR, TIM-3, and VISTA. In some embodiments, the inhibitor is an antibody against an immune checkpoint protein. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1, for example, an antibody that specifically binds to PD-1 or PD-L1. In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, ipilimumab, durvalumab, or atezolizumab. In some embodiments, the bispecific antibody or its antigen-binding fragment may be directly or indirectly linked to an additional therapeutic agent to form an antibody-drug conjugate.

[0147] Another aspect of the present invention relates to a kit comprising a bispecific antibody of the present invention or an antigen-binding fragment thereof, or a cell for producing the bispecific antibody of the present invention or an antigen-binding fragment thereof. In some embodiments, the kit may comprise a plurality of bispecific antibodies or antigen-binding fragments thereof and / or a composition comprising such antibodies or antigen-binding fragments. In some embodiments, the kit may further comprise additional activators, such as chemotherapy agents, as known to those skilled in the art. In some embodiments, the kit may further comprise reagents, buffers, containers, etc.

[0148] A method using an antibody that binds to an integrin or its antigen-binding fragment.

[0149] One aspect of the present invention relates to a method for preventing a cell from binding to fibronectin, comprising the step of contacting a cell with an effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to one or more integrins, e.g., integrin αvβ3, integrin αvβ6, and / or integrin α5β1. The method may comprise preventing the cell from binding to fibrous fibronectin, globular fibronectin, or both fibrous fibronectin and globular fibronectin. The structure and domain of fibronectin are described in Dalton & Lemmon (2021). Cells 10(9):2443, described in FIG. 1, the entire contents of which are incorporated herein by reference). Fibronectin is known to be associated with chemotherapy resistance in cancers, including cisplatin resistance. For example, Gao et al. (2016) Biochem. Biophys. Res. Commun. 476(1):35-41 and Spada et al. (2021) J. Exp. Clin. Cancer Res. Refer to 40:102. Additionally, an increase in fibronectin is known to be associated with fibrotic diseases. For example, Van Vliet et al. (2001) J. Pathol.See 193(2):256-262. Integrin subunit αv is present in various cells, including fibroblasts, epithelial cells, and neural tissue. Integrin subunits βα5 and ββ are mesenchymal-like markers, but cellular stress induces the expression of integrin subunits β3, α5, and β6 in epithelial-like cancer cells. Integrins such as α5β1, αvβ3, αvβ6, or α6β4 are observed to have increased expression in tumor-initiating cells, metastatic cells, or stress-resistant cells. In some embodiments, the method comprises the step of contacting an effective amount of the bispecific antibody of the present invention or an antigen-binding fragment thereof with a cell. In some embodiments, the method comprises the step of contacting a cell with an effective amount of the bispecific antibody of the present invention or an antigen-binding fragment thereof targeting one or more fibronectin-binding integrins, for example, prevents binding across heterogeneous tumor populations when antigens are recognized in different tumor cells; when both antigens are recognized in a single cell, increases or enhances recognition by the tumor cell, increases or enhances apoptosis of the tumor cell, and / or inhibits metastasis or growth of the tumor cell; and / or targets the tumor more effectively. In some embodiments, the tumor is a fibrotic tumor and comprises stromal cells.

[0150] In some embodiments, the method and composition disclosed herein prevent or substantially reduce the binding of fibronectin to cancer cells present at a primary tumor site by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to the binding of fibronectin to cancer cells present at a primary tumor site when the composition is not present. In some embodiments, the method and composition disclosed herein prevent or substantially reduce the binding of fibronectin to cells involved in settling, migrating, and / or infiltration into tissues by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to the binding of fibronectin to the same cells in the absence of the composition. In some embodiments, the method and composition disclosed herein prevent or substantially reduce the formation of fibronectin and collagen fibers by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to the formation of fibronectin and collagen fibers in the absence of the composition.In some embodiments, the composition of the present invention is a bispecific antibody or an antigen-binding fragment thereof described herein, said bispecific antibody or an antigen-binding fragment thereof, said bispecific antibody or an antigen-binding fragment thereof, compared to any one of the antibodies from which the bispecific antibody or an antigen-binding fragment thereof is derived, has an improvement effect of at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) in the ability to block the binding of fibronectin to cancer cells present at the primary tumor site, the ability to reduce the binding of fibronectin to cells involved in settling, migrating, and / or invading into tissues, the ability to reduce the binding of fibronectin to cells involved in fibrosis, and / or the ability to reduce the formation of fibronectin and collagen fibers.

[0151] One aspect of the present invention relates to a method for treating cancer in a subject requiring treatment for cancer expressing integrins, for example, integrin αvβ3 and / or integrin α5β1, comprising the step of administering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to one or more integrins, for example, integrin αvβ3 and / or integrin α5β1. In some embodiments, the method for treating cancer in a subject requiring treatment for cancer expressing integrin αvβ3 and / or integrin α5β1 is a method of treating cancer by administering to the subject a therapeutically effective amount of a bispecific antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising said bispecific antibody or an antigen-binding fragment thereof.

[0152] One aspect of the present invention relates to a method for treating cancer in a subject requiring treatment for cancer, comprising the following steps: a) selecting a subject having cancer cells richly expressing integrin αvβ3, integrin αvβ6 and / or integrin α5β1, and optionally immune effector cells (e.g., macrophages); and b) administering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to one or more integrins, e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1. In some embodiments, the method is a method for treating cancer comprising administering to the subject a therapeutically effective amount of a bispecific antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof. In some embodiments, step a) includes obtaining a sample of cancer from a subject and measuring the expression levels of integrins, e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1, in the sample and optionally the levels of immune effector cells.

[0153] Another aspect of the present invention relates to a method for treating cancer in a subject requiring treatment of cancer that expresses an antigen recognized by an antibody or an antigen-binding fragment thereof that specifically binds to one or more integrins, e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1, said cancer In some embodiments, a method for treating cancer in a subject requiring treatment of cancer expressing an antigen recognized by the bispecific antibody of the present invention or its antigen-binding fragment (e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1) is a method of treating cancer by administering to the subject a therapeutically effective amount of the bispecific antibody of the present invention or its antigen-binding fragment, or a pharmaceutical composition of the present invention.

[0154] Another aspect of the present invention relates to a method for treating epithelial cell carcinoma in a subject requiring treatment for epithelial cell carcinoma, wherein the method comprises the step of administering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to one or more integrins, e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1, or a pharmaceutical composition of the present invention. In some embodiments, the method for treating epithelial cell carcinoma in a subject requiring treatment for epithelial cell carcinoma is a method for treating epithelial cell carcinoma by administering to the subject a therapeutically effective amount of the bispecific antibody or an antigen-binding fragment thereof of the present invention, or a pharmaceutical composition of the present invention.

[0155] Another aspect of the present invention relates to a method for treating cancer in a subject requiring cancer treatment, comprising the following steps:

[0156] a) a step of selecting a subject rich in bone marrow-derived cells having cancer cells that express an antigen specifically bound by an antibody or an antigen-binding fragment thereof that specifically binds to one or more integrins, e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1; and

[0157] b) a step of treating cancer by administering to a subject an antibody or an antigen-binding fragment thereof that specifically binds to one or more integrins, e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1, or a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0158] In some embodiments, a method for treating cancer in a subject requiring cancer treatment includes the following steps:

[0159] a) a step of selecting a subject rich in bone marrow-derived cells having cancer cells having high expression of an antigen specifically bound by the bispecific antibody of the present invention or its antigen-binding fragment (e.g., integrin αvβ3); and

[0160] b) A step of treating cancer by administering to a subject a therapeutically effective amount of the bispecific antibody of the present invention or its antigen-binding fragment, or the pharmaceutical composition of the present invention.

[0161] The epithelial cancer may be any known type of carcinoma, such as squamous cell carcinoma, adenocarcinoma, transitional cell carcinoma, or basal cell carcinoma. In some embodiments, the cancer is a cancer associated with chronic inflammation-related fibrosis. For example, Piersma et al. (2020) Biochim. Biophys. Acta Rev. Cancer See 1873(2):188356. Examples of epithelial cancers include, but are not limited to, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, renal cancer, breast cancer, brain cancer (e.g., glioma, e.g., glioblastoma), skin cancer, gastric cancer, prostate cancer, ovarian cancer, esophageal cancer, or colorectal cancer. In some embodiments, the epithelial cancer cells are advanced epithelial cancer cells. In some embodiments, the epithelial cancer cells have at least partially transitioned to mesenchymal cells and, for example, express one or more mesenchymal antigens. In certain embodiments, the cancer cells exhibit resistance or refractoryness to chemotherapy, which may be attributed to epithelial-mesenchymal transition.

[0162] In some embodiments, the method may further comprise the steps of isolating bone marrow-derived cells from a subject, contacting the bone marrow-derived cells with one or more integrins, e.g., an antibody or an antigen-binding fragment thereof that specifically binds to integrin αvβ3, integrin αvβ6 and / or integrin α5β1, optionally a bispecific antibody or an antigen-binding fragment thereof, or a pharmaceutical composition, and administering the contacted bone marrow-derived cells to the subject.

[0163] In some embodiments, the cancer is epithelial cancer, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, renal cancer, breast cancer, brain cancer (e.g., glioma, e.g., glioblastoma), skin cancer, gastric cancer, prostate cancer, esophageal cancer, or colorectal cancer. The epithelial cancer may be any known type of cancer. Examples of epithelial cancer include, but are not limited to, cancer of the gastrointestinal tract, breast, lung (e.g., non-small cell lung cancer), colon, prostate, or bladder. In some embodiments, the epithelial cancer cells are terminal epithelial cancer cells. As used herein, terminal or advanced stage refers to cancer of stage III or IV according to the TNM staging system. In some embodiments, the epithelial cancer cells have at least partially metastasized to mesenchymal cells and express, for example, one or more mesenchymal antigens. Cancer-associated fibroblasts, macrophages, and other immune cells secrete various cytokines and factors that act on tumor cells to activate the expression of transcription factors that induce EMT. Mesenchymal-like carcinoma cells also alter the immune components of the tumor to an immunodeficient state that excludes anti-tumor immune cell types and recruits tumor-promoting macrophages. In certain embodiments, the epithelial cancer cells exhibit resistance or refractoryness to chemotherapy, which may be attributed to epithelial-mesenchymal transition. In some embodiments, the cancer has a KRAS mutation (e.g., Huang et al. (2021) Signal Transduction and Targeted Therapy See 6:386) or EGFR mutations (e.g., Sigismund et al. (2018) Molecular Oncology Includes 12(1): See 3-20).

[0164] In some embodiments, two or more antibodies or their antigen-binding fragments, for example, bispecific antibodies or their antigen-binding fragments, that specifically bind to one or more integrins, for example, integrin αvβ3, integrin αvβ6 and / or integrin α5β1, may be delivered to a target. In some embodiments, antibodies or their antigen-binding fragments that specifically bind to one or more integrins, for example, integrin αvβ3, integrin αvβ6 and / or integrin α5β1, may bind to multiple target antigens.

[0165] Another aspect of the present invention relates to a method for treating a fibrotic disease by administering to a subject in a therapeutically effective amount a pharmaceutical composition comprising an antibody or its antigen-binding fragment that specifically binds to one or more integrins, for example, integrin αvβ3, integrin αvβ6 and / or integrin α5β1, an antibody or its antigen-binding fragment that specifically binds to one or more integrins, for example, integrin αvβ3, integrin αvβ6 and / or integrin α5β1, optionally a bispecific antibody or its antigen-binding fragment of the present invention, or an antibody or its antigen-binding fragment that specifically binds to one or more integrins, for example, integrin αvβ3 and / or integrin α5β1, to the subject in need of treatment for the fibrotic disease. In some embodiments, the fibrotic disease is ocular fibrosis, cardiac fibrosis, hepatic fibrosis, intestinal fibrosis, pulmonary fibrosis, pancreatic fibrosis, renal fibrosis, non-alcoholic fatty liver disease, systemic sclerosis, idiopathic pulmonary fibrosis, fibromyalgia, keloid, or cutaneous fibrosis. In one embodiment, the cell is a mesenchymal cell, a fibroblast, or a macrophage.

[0166] Another aspect of the present invention comprises a method for inhibiting collagen organization in a subject, the method comprising the step of administering a composition targeting fibronectin. In some embodiments, the composition targets one or more fibronectin receptors. For example, the composition targets two or more fibronectin receptors. In some embodiments, the composition inhibits the expression and / or function of one or more fibronectin receptors. In some embodiments, inhibiting the expression and / or function of one or more fibronectin receptors inhibits collagen organization but does not affect cell adhesion.

[0167] Another aspect of the present invention relates to a method for reversing collagen organization in a subject, comprising the step of administering a composition targeting fibronectin. In some embodiments, the composition targets one or more fibronectin receptors. For example, the composition targets two or more fibronectin receptors. In some embodiments, the composition inhibits the expression and / or function of one or more fibronectin receptors.

[0168] In some embodiments, the composition comprises an antisense oligonucleotide, RNAi, or siRNA. In some embodiments, the composition comprises one or more antisense oligonucleotides, RNAi, or siRNA. In some embodiments, the composition targets an integrin, e.g., a heterodimer integrin. In some embodiments, the antisense oligonucleotide, RNAi, or siRNA targets a specific integrin receptor subunit. In some embodiments, the target is a single integrin receptor or a combination comprising integrin αvβ3 and / or integrin α5β1.

[0169] RNAi therapeutics comprise polynucleotides complementary to a portion of the target sequence mRNA and generally have a size range of 15 to about 50 base pairs. In one exemplary embodiment, siRNA is a nucleic acid capable of forming double-stranded RNA having the ability to reduce or inhibit the expression of a gene or target gene, wherein each complementary sequence of the double-stranded siRNA has a length of about 15 to about 50 nucleotides, and the double-stranded siRNA has a length of about 15 to about 50 base pairs. Additionally, the use of small hairpin RNA (shRNA) is also being considered. shRNA comprises an antisense strand consisting of about 19 to about 25 nucleotides, a short nucleotide loop (about 5 nt to 9 nt) located thereafter, and a corresponding sense strand located thereafter. In one embodiment, RNAi is microRNA or miRNA, i.e., endogenous RNA, some of which are known to regulate the expression of protein-coding genes at a post-transcriptional level. For example, Lim et al. (2003) Science 299:1540; Lee & Ambros (2001) Science 294:862; Lau et al. (2001) Science See 294:858-861; Lagos-Quintana et al. (2003) RNA 9:175-179.

[0170] There are various criteria for selecting the nucleic acid to use, which may include scanning the mRNA sequence of the target, for example, Sui et al. (2002) Proc. Natl. Acad. Sci.It may include empirical determinations according to USA 99:5515-20) and may include confirming that the sequence does not have significant sequence homology with other genes as analyzed by BLAST search. Additional approaches may include methods in which any accessible site in endogenous mRNA can be targeted to be degraded by a synthetic oligodeoxyribonucleotide / RNase H method (e.g., Lee et al. (2002) Nature Biotechnol. (See 20:500-05). The RNAi treatment may include miRNA or siRNA, or pre-miRNA that is treated by Dicer to form miRNA. Additionally, the RNAi treatment may include dsRNA or shRNA that is treated by Dicer to form siRNA. The polynucleotide may include one or more modifications to suppress innate immune activation, enhance activity and specificity, and reduce off-target toxicity. Exemplary examples are, for instance, Provost et al. (2002). EMBO J. 21(21):5864-5874; Tabara et al. (2002) Cell 109(7):861-871; Martinez et al. (2002) Cell 110(5):563; and Hutvagner & Zamore (2002) ScienceIt can be found in 297:2056. In certain embodiments, the single-stranded RNAi formulations disclosed herein may include substitutions or modifications comprising chemically modified nucleotides and non-nucleotides that may be introduced into the backbone, sugar, base, or nucleotide. The use of substituted or modified single-stranded RNAi formulations may be designed to have an increased half-life within the target. Additionally, specific substitutions or modifications may be used to improve the bioavailability of single-stranded RNAi formulations by targeting specific cells or tissues or by enhancing the intracellular uptake of the single-stranded RNAi formulations. Exemplary modifications and locations within RNAi polynucleotides are described in Hu et al. (2020). Signal Transduct. Targeted Ther. 5:100, incorporated herein by reference, for example, see FIG. 2 and FIG. 3, in particular description regarding variations) is described.

[0171] In some embodiments, the composition comprises an antibody or an antigen-binding fragment thereof that specifically binds to fibronectin. In some embodiments, the antibody or the antigen-binding fragment thereof specifically binds to one or more integrin subunits, for example, alpha and / or beta subunits. In some embodiments, the antibody or the antigen-binding fragment thereof is monospecific, or the antibody or the antigen-binding fragment thereof is a bispecific antibody or its antigen-binding fragment as described in detail in other parts of this specification, and binds to an integrin selected from, for example, integrin αvβ3, integrin αvβ6, and integrin α5β1.

[0172] Another aspect of the present invention relates to a method for inhibiting the expression of Yes-associated protein 1 (YAP) or a YAP target gene in a subject, comprising the step of administering to a subject an antibody or its antigen-binding fragment that specifically binds to one or more integrins, e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1, optionally a bispecific antibody or its antigen-binding fragment of the present invention, a polynucleotide encoding one or more integrins, e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1, an antibody or its antigen-binding fragment that specifically binds to one or more integrins, e.g., integrin αvβ6 and / or integrin α5β1, optionally a bispecific antibody or its antigen-binding fragment of the present invention, or a vector comprising said polynucleotide. In some embodiments, the expression of YAP or the expression of a YAP target gene is inhibited by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to the expression of YAP or the YAP target gene in the absence of an antibody or its antigen-binding fragment. In one embodiment, the YAP target gene is CTGF or CYR61. For example, Pobbati et al. (2020) Theranostics Refer to 10(8):3622-3635). In some embodiments, inhibiting the expression of the YAP protein and / or the YAP target gene reduces solid tumor growth, inhibits cancer, and reduces oncogenic processes such as stemness, drug resistance, and immunosuppression by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to solid tumor growth, cancer growth, or stemness, drug resistance, and immunosuppression in the absence of the antibody or its antigen-binding fragment.

[0173] The method of the present invention may further include the step of administering an additional therapeutic agent or treatment (e.g., surgery, radiation therapy) to the subject. The fibrosis therapeutic agent comprises antifibrotic agents, e.g., nintedanib, pirfenidone, sarcatinib, FG-3019, bardoxolone methyl, afabetalone, angiotensin-converting enzyme (ACE) inhibitors, and angiotensin II receptor blockers (ARBs). The cancer therapeutic agent comprises, without limitation, 1) vinca alkaloids (e.g., vinblastine, vincristine); 2) epipodophyllotoxins (e.g., etoposide and tenifoside); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mitramycin), and mitomycin (mitomycin C)); 4) Enzymes (e.g., L-asparaginase); 5) Biological response modulators (e.g., interferon-alpha); 6) Platinum coordination complexes (e.g., cisplatin and carboplatin); 7) Anthracendiones (e.g., mitoxantrone); 8) Substituted elements (e.g., hydroxyurea); 9) Methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MIH)); 10) Adrenocorticosteroids (e.g., mitothan (o,p'-DDD) and aminoglutethimide); 11) Adrenocortical steroids (e.g., prednisone); 12) Progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and ethinyl estradiol); 14) anti-estrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone propionate and fluoxymesterone); 16) anti-androgens (e.g., flutamide); and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide).Other cancer treatments, without limitation, include anti-angiogenic agents, e.g., antibodies against VEGF such as bevacizumab (Avastin®) and ranibizumab (Lucentis®), and other angiogenesis-promoting factors (e.g., bFGF, angiopoietin-1), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alpha conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurine, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation (Abraxane®), soy isoflavone (genistein), tamoxifen citrate, thalidomide, ADH-1 (Exherin), AG-013736, AMG-706, AZD2171, and sorafenib Includes tosylate, BMS-582664, CHIR-265, pazopanib, PI-88, batalanib, everolimus, suramine, sunitinib maleate, XL184, ZD6474, ATN-161, silengitide, celecoxib, and methotrexate.

[0174] In some embodiments, the method further comprises the step of administering a CD47 blocker to a subject to enhance the phagocytosis of cancer cells. Such agents include CD47 blocking monoclonal antibodies (e.g., Hu5F9-G4, CC-90002, Ti-061, or SRF231) or SIRPα-Fc fusion proteins (e.g., TTI-621, TTI-622, ALX148). However, one of the advantages of the present invention is that the method is effective against cancer regardless of whether the cancer cells express CD47. Accordingly, in some embodiments, the method of the present invention is used to treat cancer that expresses CD47. In some embodiments, the method of the present invention is used to treat cancer that does not express CD47. In some embodiments, the method of the present invention does not include the step of administering a CD47 blocker to a subject.

[0175] In some embodiments, the method further comprises the step of administering an immune checkpoint inhibitor to a subject. Immune checkpoints are well known in the art and include, without limitation, PD-1, PD-L1, PD-L2, CTLA4, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, A2AR, TIM-3, and VISTA. In some embodiments, the inhibitor is an antibody against an immune checkpoint protein. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4 which is abundantly expressed in mesenchymal tumors, and is, for example, an antibody that specifically binds to PD-1, PD-L1, or CTLA-4. In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, ipilimumab, durvalumab, or atezolizumab.

[0176] In some embodiments, the method further comprises the step of administering an EGFR inhibitor to a subject. Such agents include tyrosine kinase inhibitors (e.g., erlotinib, gefitinib, lapatinib, osimertinib, neratinib) and monoclonal antibodies (e.g., cetuximab, necitumumab, panitumumab). In some embodiments, the method further comprises the step of administering a KRAS inhibitor to a subject, and in particular for patients having a KRAS G12C mutation, the KRAS inhibitor may be sotorasib, MRTX1133, or ARS1620.

[0177] In certain embodiments, one or more integrins used in the method of the present invention, for example, integrin αvβ3, integrin αvβ6 and / or integrin α5β1, an antibody or its antigen-binding fragment that specifically binds to them, optionally a bispecific antibody or its antigen-binding fragment, is administered directly to a subject. In some embodiments, one or more integrins, for example, integrin αvβ3, integrin αvβ6 and / or integrin α5β1, an antibody or its antigen-binding fragment that specifically binds to them is suspended in a pharmaceutically acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or administered subcutaneously, intramuscularly, intrathecally, intraperitoneally, rectalally, vaginally, nasally, gastricly, tracheally, or lungly. In other embodiments, intratracheal or intrapulmonary administration may be performed using a standard nebulizer, jet nebulizer, wire mesh nebulizer, dry powder inhaler, or metered-dose inhaler. The formulation may be delivered directly to the site of disease or disorder, such as the lungs, kidneys, or intestines, for example, in situ ( in situIt may be administered via ). The required dose depends on the choice of administration route, characteristics of the formulation, characteristics of the patient's disease, the subject's size, body weight, body surface area, age and sex, other drugs administered concomitantly, and the judgment of the attending physician. The appropriate dose for each formulation is in the range of 0.01 to 100 μg / kg. Given the variety of available formulations and the different efficiencies of various administration routes, the required dose may vary over a wide range. For example, oral administration is expected to require a higher dose than administration by intravenous (iv) injection. These variations in dose levels can be adjusted using standard empirical methods for optimization, as is well known in the art. Administration may be performed once or multiple times (e.g., 2, 3, 4, 6, 8, 10, 20, 50, 100, 150 or more times). Encapsulating the compound in a suitable carrier (e.g., polymer microparticles or nanoparticles or implantable devices) can increase delivery efficiency, especially when administered orally.

[0178] "Pharmaceuticalally acceptable" means a substance that is not biologically or otherwise undesirable; that is, a substance that can be administered to a subject without causing undesirable biological effects, such as toxicity.

[0179] The formulation of the present invention may optionally include a medicinal agent, a pharmaceutical preparation, a carrier, an adjuvant, a dispersant, a diluent, etc.

[0180] An antibody or its antigen-binding fragment that specifically binds to one or more integrins of the present invention, e.g., integrin αvβ3, integrin αvβ6 and / or integrin α5β1, and optionally a bispecific antibody or its antigen-binding fragment, may be prepared into a formulation for administration in a pharmaceutical carrier according to known techniques. For example, Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, PA) may be referenced. When preparing a pharmaceutical formulation according to the present invention, the formulation is mixed with components including a generally acceptable carrier. The carrier may be solid or liquid, or a combination thereof, and may be formulated together with the formulation in the form of a unit dose formulation, e.g., a capsule or a vial, and the unit dose formulation may contain 0.01% or 0.5% to 95% or 99% of the formulation by weight. One or more formulations may be included in the formulation of the present invention, and the formulation may be prepared by any one of the pharmaceutical manufacturing techniques well known in the art.

[0181] The formulations of the present invention include formulations suitable for oral, rectal, topical, buccal (e.g., sublingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragmatic muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical (i.e., both skin and mucosal surfaces including the airway surface), nasal, transdermal, intra-articular, intrathecal, and inhalation administration. Additionally, formulations suitable for administration to the liver via portal vein delivery, direct injection into an organ (e.g., intrahepatic, intracerebral or pancreatic injection for delivery to the central nervous system), or intra-body cavity injection are also included. In any case, the most suitable route will depend on the nature and severity of the disease to be treated and the characteristics of the specific agent used.

[0182] For injectable use, the carrier is generally a liquid such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline, bacteriostatic water, or Cremophor EL® (BASF, Parsippany, NJ). For other administration methods, the carrier may be a solid or a liquid.

[0183] For oral administration, the formulation may be administered in solid formulations such as capsules, tablets, and powders, or in liquid formulations such as elixirs, syrups, and suspensions. The formulation may be encapsulated in gelatin capsules with a powder carrier and inert ingredients such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, etc. Examples of additional inert ingredients that may be added to impart color, taste, stability, buffering capacity, dispersibility, or other known desirable properties include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar diluents may also be used to manufacture compressed tablets. Both tablets and capsules may be manufactured as sustained-release formulations to ensure continuous release of the drug over several hours. Compressed tablets may be coated with sugar or film to mask an unpleasant taste and protect the tablet from the external environment, or they may be enteric-coated to selectively disintegrate in the gastrointestinal tract. Liquid formulations for oral administration may include coloring agents and flavoring agents to enhance patient solubility.

[0184] Formulations suitable for oral (sublingual) administration generally include lozenges containing a formulation in a base with added flavor, such as sucrose and acacia or tragacanth, and pastiles containing a formulation in an inert base, such as gelatin and glycerin or sucrose and acacia.

[0185] The formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injectable solutions of the drug, and it is preferable that these formulations be isotonic with the blood of the intended recipient. These formulations may include antioxidants, buffers, antimicrobial agents, and solutes that maintain isotonicity with the blood of the transfusion recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickeners. The formulations may be provided in the form of unit-dose or multi-dose containers, e.g., sealed ampoules and vials, and may be stored in a lyophilized state, requiring only the addition of a sterile liquid carrier, such as physiological saline or water for injection, immediately before use.

[0186] Injectable solutions and suspensions prepared on the spot may be prepared from sterile powders, granules, and tablets of the types described above. For example, in one aspect of the invention, an injectable, stable, and sterile composition is provided that comprises a formulation of the invention in the form of a unit dose in a sealed container. The formulation is provided in the form of a lyophilized material and can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dose form generally comprises about 1 mg to about 10 g of said formulation. If the formulation is substantially insoluble in water, the formulation may be emulsified in an aqueous carrier using a sufficient amount of a pharmaceutically acceptable emulsifier. One such useful emulsifier is phosphatidylcholine.

[0187] A formulation suitable for rectal administration is preferably provided in the form of a unit dose suppository. Such suppositories may be manufactured by mixing the formulation with one or more conventional solid carriers, such as cocoa butter, for example, and then molding the resulting mixture.

[0188] Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that may be used include petroleum jelly, lanolin, polyethylene glycol, alcohol, transdermal absorption promoters, and combinations of two or more of these.

[0189] Formulations suitable for transdermal administration can be provided as individual patches configured to be in close contact with the recipient's epidermis for an extended period. Formulations suitable for transdermal administration can also be delivered via iontophoresis (e.g., Tyle (19886) Pharm. Res. (See 3:318), generally takes the form of a buffered aqueous solution of the compound. Suitable formulations include citric acid or bis / tris buffer (pH 6) or an ethanol / water solution, and the concentration of the compound is 0.1 to 0.2 M.

[0190] The above formulation may alternatively be formulated for nasal administration or otherwise administered to the subject's lungs by any suitable means. For example, it may be administered by having the subject inhale an aerosol suspension of inhalable particles containing the above formulation. The inhalable particles may be liquid or solid. The term "aerosol" includes all forms suspended in a gas that can be inhaled into the bronchioles or nasal cavity. Specifically, an aerosol includes a suspension of droplets in a gas, such as those that may be produced by a metered-dose inhaler, nebulizer, or mist sprayer. An aerosol also includes a dry powder composition suspended in air or another carrier gas, which may be delivered, for example, by blowing from an inhaler device. Ganderton & Jones (1987) Drug Delivery to the Respiratory Tract, Ellis Horwood; Gonda (1990) Crit. Rev. Therapeut. Drug Carrier Systems 6:273-313; and Raeburn et al. (1992) J. Pharmacol. Toxicol. Meth. 27:143.Refer to [the following]. An aerosol of liquid particles containing a formulation may be generated by any suitable means, such as a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those skilled in the art. For example, refer to US Patent No. 4,501,729. Likewise, an aerosol of solid particles containing a formulation may be generated using a solid particle medicine aerosol generator according to techniques known in the pharmaceutical field.

[0191] Alternatively, the compound may be administered topically rather than systemically, for example, as a sustained-release or extended-release formulation.

[0192] In addition, the present invention provides liposomal formulations of the formulations and salts thereof disclosed herein. Techniques for preparing liposomal suspensions are well known in the art. If the compound or its salt is a water-soluble salt, it can be incorporated into lipid vesicles using conventional liposome techniques. In such cases, since the formulation is water-soluble, it is substantially captured within the hydrophilic core or nucleus of the liposome. The lipid layer used may have a general composition and may or may not contain cholesterol. If the compound or salt of interest is insoluble in water, conventional liposome formation techniques can be used to encapsulate a significant amount of the salt within a hydrophobic lipid bilayer forming the liposome structure. In both cases, the size of the resulting liposomes can be reduced using standard sonication and homogenization techniques.

[0193] A liposome formulation containing a formulation can be freeze-dried to produce a freeze-dried product, and said freeze-dried product is water, etc. It can be reconstituted into a pharmaceutically acceptable carrier to re-form a liposome suspension.

[0194] In the case of water-insoluble substances, pharmaceutical compositions containing water-insoluble substances, such as aqueous base emulsions, can be prepared. In such cases, the composition contains a sufficient amount of pharmaceutically acceptable emulsifier to emulsify the desired amount of emulsifier. Particularly useful emulsifiers include phosphatidylcholine and lecithin.

[0195] In a specific embodiment, the compound is administered to a subject at a therapeutically effective dose as previously defined. The dose of the pharmacologically active agent may be determined by methods known in the art, for example Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA) may be referenced. The therapeutically effective dose of a specific formulation will vary somewhat depending on the formulation and the subject, and will depend on the subject's condition and route of administration. Generally, doses of about 0.1 to about 50 mg / kg produce a therapeutic effect, and all doses are calculated based on the weight of the formulation. Due to toxicity issues at high doses, intravenous doses may be limited to lower levels, such as about 10 mg / kg or less, and all doses are calculated based on the weight of the formulation. For oral administration, doses of about 10 mg / kg to about 50 mg / kg may be used. Generally, for intramuscular injection, doses of about 0.5 mg / kg to about 5 mg / kg may be used. Specific doses are about 1 μmol / kg to about 50 μmol / kg based on the weight of the formulation, and more specifically, about 22 μmol / kg and about 33 μmol / kg for intravenous and oral administration, respectively.

[0196] In a specific embodiment of the present invention, one or more administrations (e.g., two, three, four or more administrations) may be performed over various time intervals (e.g., hourly, daily, weekly, monthly, etc.) to obtain a therapeutic effect.

[0197] The present invention may be used in veterinary and medical applications. Suitable subjects include both birds and mammals, with mammals being preferred. As used herein, the term "mammal" includes, but is not limited to, humans, primates, cattle, sheep, goats, horses, cats, dogs, lagomorphs, etc. Human subjects include newborns, infants, adolescents, and adults. Subjects may be persons requiring the method of the present invention, for example, persons who have cancer or are suspected of having cancer. Subjects may be experimental animals, for example, animal models of diseases.

[0198] The present invention is explained more specifically through the following examples, and since various modifications and variations will be obvious to those skilled in the art, the following examples are for illustrative purposes only.

[0199] Examples

[0200] Example 1: Material and Method

[0201] Reagents, chemicals, and commercial antibodies.The primary antibodies used in the present invention are FN (E5H6X)(Cell Signaling #26836, RRID: AB_2924220, 1:1000 for Western blot, 1:200 for immunohistochemistry), FN (DH1)(Novus Biologicals NBP1-51723, RRID: AB_11059914, 1:200 for immunofluorescence staining), COL1A1 (E8F4L)(Cell Signaling #72026, RRID: AB_2904565, 1:1000 for Western blot, 1:200 for immunofluorescence staining), CTGF (D8Z8U)(Cell Signaling #86641, RRID: AB_2800085, 1:1000 for Western blot), vinculin(Boster #MA1103, RRID: It includes AB_3082541, 1:15,000 for Western blot), GAPDH (D16H11) (Cell Signaling #5174, RRID: AB_10622025, 1:3000 for Western blot), anti-α5 integrin antibody (P1D6) (EMB Millipore MAB1956Z, RRID: AB_94455, 10 μg / ml for flow cytometry), and CD31 (R&D Systems #AF3628, RRID: AB_2161028, 1:40 for immunohistochemistry). The anti-αvβ integrin antibody (LM609) (10 μg / ml for flow cytometry) was purchased from Millipore (MAB1976, RRID: AB_2296419). The pre-designed siRNA used in the present invention was purchased from Millipore Sigma. Each siRNA combination consists of two different types of siRNA (siRNA1 and siRNA2, a 1:1 mixture) targeting different gene regions of the gene of interest. The IDs of each siRNA are presented in Table 1.

[0202]

[0203] New antibody.ABT101 (heavy chain ETAR.VH-h4.HC.S228P, SEQ NO. 7; and light chain ETAR.VH-hk.LC, SEQ NO. 8) and ABT701 (heavy chain VH-h4.HC.S228P, SEQ NO. 5; and light chain VL-hk.LC, SEQ NO. 6) are hIgG4-S228P monoclonal antibodies that recognize human integrins αvβ3 and α5β1, respectively (Table 2). ABT601 is a bispecific antibody designed to dually recognize fibronectin (FN)-binding integrins αvβ and α5β. ABT601 consists of an hIgG4-S228P Fc domain identical to the antigen-recognizing Fab domain of ABT101 and ABT701.

[0204]

[0205] cell. PANC1 human pancreatic cancer cells were obtained from the American Type Culture Collection (ATCC CRL-1469, RRID: CVCL_0480) and cultured in DMEM. Cell line authentication via Short Repeat Sequence (STR) analysis was performed on PANC1 cells in April 2015 and November 2024. KP4 human pancreatic cancer cells were obtained from the RIKEN BioResource Research Center Cell Bank (RRID: CVCL_1338) in January 2023 with STR cell line authentication and cultured in RPMI. Dr. Andrew Lowy (UC San Diego) [referenced] previously established (Hurtado de Mendoza et al., (2021) from fresh surgical specimens of human PDAC tissue Nat. Commun. (12(1):1541) Low-passage stock vials of the immortalized CAF cell lines hPCF1299 (CAF-1299) and hPCF1424 (CAF-1424) were provided in June 2022. All cells were proliferated after provision, and M. mycoidesAfter confirming the presence of mycoplasma contamination using PCR to detect the 16S rRNA gene of the group (forward primer: 5'-CGA AAG CGG CTT ACT GGC TTG TT-3' (SEQ No. 9); reverse primer: 5'-TTG AGA TTA GCT CCC CTTCAC AG-3' (SEQ No. 10)), the cells were cryopreserved at a low passage (Bascunana et al., (1994) J. Bacteriol. 176(9):2577-86).

[0206] Generation of cell-free ECM and cell-based analysis. To allow cells to produce and deposit sufficient extracellular matrix (ECM), cells were seeded on 8-well chamber slides, glass coverslips, or tissue-treated cell culture plates for up to 7 days. Decellularization was performed according to a previously reported method (Harris et al., (2018) Methods Cell. Biol. 143:97-114). Samples were washed twice with Hanks' Balanced Salt Solution (HBSS) and then incubated in lysis buffer (8 mM Na2HPO4, 1% NP-40, pH 9.6) at 37°C for 15–20 minutes. Subsequently, they were washed three times with wash buffer (10 mM Na2HPO4, 300 mM KCl, pH 7.5), washed again three times with sterile deionized water, and stored in HBSS. The cell-removed ECM thus prepared was used for immunofluorescence staining or other cell-based assays. For cell-based analysis, pancreatic cancer cells were seeded onto cell-removed ECM in the presence of a medium containing 10% or 2% FBS and cultured for 24 hours, after which the cells were recovered for Western blot analysis.

[0207] Quantitative RT-PCRRNA was isolated using the RNeasy RNA Purification Kit (Qiagen, 75144) according to the manufacturer's instructions. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814), and RT-PCR was performed using a CFX96 (Bio-Rad) instrument with SYBR®Green dye (Bio-Rad, 1725272). The nucleotide sequences of the custom primers are presented in Table 3.

[0208]

[0209] Mouse experiment approved. All experiments using mice were conducted in accordance with Protocol S05018, which was approved by the Institutional Animal Care and Use Committee (IACUC) of UC San Diego. All experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All laboratory animals were housed under standard rearing conditions; that is, they were provided with free access to food and water, housed in standard rearing cages, and maintained in an environment with controlled temperature and light-dark cycles. In vivo ( in vivo The cell line used in the experiment was confirmed to be negative for a panel of human-derived pathogens.

[0210] Subcutaneous xenograft model The number of PANC1 cells injected varies significantly from study to study (Kuninty et al., (2019) Sci. Adv. 5(9):eaax2770; Watabe et al., (2020) J. Nucl. Med. 61(4):563-569; Wang et al., (2014) Pancreas43(2):291-297), this is considered to be due to the use of various strains of immunodeficient mice. Since nu / nu mice have a relatively lower degree of immune system impairment than NOD-SCID or NSG strain mice, in the experiment of Fig. 17A, 5×10 5 500,000 cells were selected as the injection volume. 5×10 5 Canine human PANC1 cells were transfected with different siRNAs for 72 hours, with the same number (5×10⁶) 5 Human CAF-1299 cells were mixed or not mixed with (dog) cells. Gene expression inhibition was confirmed via Western blot.

[0211] For the antibody-treated group, PANC1 or CAF-1299 cells were pre-mixed with 10 μg / mL of antibody for 10 minutes prior to injection. Cells were suspended in a 1:1 mixture of HBSS and phenol red-free basement membrane matrix (BD Biosciences) (total volume 100 μL per injection) and injected subcutaneously into 6–8-week-old female immunodeficient nu / nu mice (Charles River Labs #088, RRID: IMSR_CRL:088). Fresh antibody (10 mg / kg) was injected intraperitoneally twice a week, and mice were observed twice a week to check for the development of palpable tumors. Tumors were counted as tumors if their volume exceeded 100 mm³.

[0212] Orthopedic pancreatic cancer model. To determine the appropriate number of cells to inject that can evaluate changes in tumor initiation at a relatively early stage, 5×10 6 Pieces, 1×10 6 Pieces or 0.2×10 6Preliminary experiments were performed on the orthotopic transplantation of canine KP4-luc cells into the pancreas. At 5 weeks post-injection, no tumors were detected by bioluminescence imaging, but at 10 weeks, 5×10 6 Dog or 1×10 6 In the group injected with 10 KP4-Luc cells, tumors were detected in all 3 / 3 mice, whereas 0.2×10 6 In the group injected with dog cells, tumors were detected in only 1 out of 3 animals (1 / 3). Accordingly, 1×10 6 It was determined that detectable tumors would form in all mice within 5–10 weeks upon orthotopic transplantation of canine KP4-Luc cells into the pancreas; furthermore, it was anticipated that co-injection with CAF would further promote tumor development, resulting in detectable tumors even before the 5-week mark. In the experiment shown in Fig. 13A, 1×10⁶ mice stably transduced with luciferase lentivirus were used. 6 The same number (1×10⁶) of human KP4 cells (KP4-Luc) transfected using different siRNAs for 72 hours 6 Human CAF-1299 cells were mixed with or without mixing. Gene expression inhibition was confirmed by Western blot. After suspending cells in HBSS, they were injected into the pancreas of 6 to 8-week-old female immunodeficient nu / nu mice (Charles River Labs #088, RRID: IMSR_CRL:088). Tumor growth was monitored twice a week using non-invasive bioluminescence imaging with an IVIS Spectrum system (Perkin-Elmer). Images were acquired from all mice 10 minutes after injection of D-luciferin (L9504, Sigma-Aldrich). Immunoblot analysis to evaluate the duration of siRNA-mediated gene expression inhibition for FN1 in CAFs showed that inhibition of FN expression was effectively maintained until day 6 of culture, but decreased significantly on day 14.

[0213] Immunofluorescence staining and confocal microscopy. CAFs were seeded onto 8-well chamber slides (Nunc Lab-Tek Chamber Slide, Thermo Scientific) and cultured overnight. The following day, cells were treated with siRNA or antibodies (10 μg / mL) targeting the gene of interest for 72 hours, followed by fixation in 4% paraformaldehyde at room temperature for 15 minutes. For targeting pre-existing substrates, CAFs were seeded and cultured for 72 hours to allow substrate deposition. After 72 hours, antibodies (10 μg / mL) were added, and the cells were incubated for an additional 72 hours. Subsequently, the cells and substrates were fixed in 4% paraformaldehyde at room temperature for 15 minutes. In all cases, the cells were then incubated for 45 minutes in blocking buffer containing HBSS or HBSS-Tween®20 (0.2%) supplemented with 5% horse serum. Subsequently, cells were incubated overnight at 4°C with a primary antibody (anti-FN or COL1A1 antibody) diluted 1:200, followed by incubation with a secondary antibody diluted 1:1000 for 1 hour, and stained with DAPI for 5 minutes at room temperature. Images were acquired as multiple Z-stack images using a Nikon Eclipse Ti C2 confocal microscope and analyzed using NIS-Elements Viewer 5.21. Fluorescence signals were quantified as area fraction (%) using ImageJ.

[0214] Immunoblot. Immunoblotting is a previously reported method (Efthymiou et al., (2020) Front. Oncol.The procedure was performed according to 10:641. Specifically, cells were washed twice with HBSS and then lysed using 1× RIPA buffer containing protease and phosphatase inhibitors, 2× sample buffer containing 1× reducing agent, or, in the case of conditioned culture supernatant, 4× sample buffer containing 1× reducing agent (Bio-Rad #1610737 and #1610747). Subsequently, BCA analysis (Thermo, 23227) was performed to measure protein concentration, and the protein concentration of the lysate was equalized. Sample buffer (NuPAGE™LDS Sample Buffer 4X, Sigma #NP0007) and a reducing agent (NuPAGE™Sample Reducing Agent, Sigma #NP0009) were added to the cell lysate. All samples were heated at 95°C for 5 minutes. 30 μL of each sample containing 10 μg of protein or Laemmli buffer was loaded onto an SDS-PAGE gel. Blocking was performed by adding 5% BSA to TBS-T, and the antibody reaction was also performed by adding 5% BSA to TBS-T buffer.

[0215] Flow Cytometry The cell pellet was washed with PBS and blocked with a PBS solution containing 1% BSA for 30 minutes at room temperature. It was then stained with a designated primary antibody or unstained using a fluorescently labeled secondary antibody. Cells were cultured with the Live / Dead Fixable Blue Dead Cell Stain Kit (Invitrogen, L23105). Flow cytometry was performed using a BD Fortessa X-20 (BD) analyzer, and the data were analyzed using FlowJo (Treestar) software.

[0216] Immunohistochemistry.Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections using the ImmPRESS®Excel Staining Kit (Vector, MP-7602) according to the manufacturer's instructions. For human FN integrin, low pH antigen recovery (Invitrogen, 00-4955-58) was performed at 95°C for 40 minutes. For CD31, high pH antigen recovery (Invitrogen, 00-4956-58) was performed at 95°C for 40 minutes. Trichrome staining was performed using Abcam AB150686. Slides were imaged at the UCSD School of Medicine Microscopy Facility using an Olympus VS200 Slide Scanner. Scanned images were analyzed using QuPath Open Software for Bioimage Analysis (Spada et al., (2021) J. Exp. Clin. Cancer Res. Protein expression was analyzed by calculating the area fraction of protein expression per tumor tissue area (40(1):102).

[0217] Atomic Force Microscope (AFM). AFM measurement is MFP-3D It was performed using an atomic force microscope (Asylum Research). The steady-state spring constant is 0.08 N·m -1A silicon nitride cantilever (Nano World, PNP-TR-50) with a length of 200 μm was used. The cantilever was calibrated using the thermal fluctuation method, and the calibration results were verified by probing with glass of known elasticity. The sample used was an OCT-embedded frozen human PDAC tissue section with a thickness of 20 μm; after thawing, it was immersed in HBSS for 5 minutes to equilibrate at room temperature. An indentation test on the sample was performed at a loading rate of 2 μm per second to generate 16 force curves in a uniformly distributed area of ​​20 μm × 20 μm. The Young's modulus of the sample was determined by fitting the force curves using a Hertz model with a Poisson ratio set to 0.5.

[0218] Example 2: Blocking fibronectin binding in cancer-associated fibroblasts

[0219] After treating CAF1299 cancer-associated fibroblasts with siRNA for 72 hours, immunostaining for fibronectin and collagen I was performed as shown in Fig. 1. The Western blot in Fig. 1B shows the effects of different siRNAs or integrin antibodies on the protein expression levels of FN, collagen I, α5, and β3 in CAF1299, and demonstrates that α5 and β3 are required for the assembly of fibronectin and collagen I substrates.

[0220] CAF1299 cancer-associated fibroblasts were treated for 72 hours with 10 μg / mL of the monovalent antibodies anti-αvβ3 (ABT101) or anti-α5β1 (ABT701), or the bispecific antibody of the example anti-αvβ3α5β1 (ABT601; Fig. 2A), and then immunostained for fibronectin and collagen I (Fig. 2B). In addition, Western blots were performed to determine the effects of different integrin antibodies on the protein levels of FN, collagen I, and GAPDH in CAF1299, and it was demonstrated that antibodies targeting integrins α5β1 and αvβ3 significantly inhibited the assembly of fibronectin and collagen I substrates (Figs. 2C and 3).

[0221] Quantitative RT-PCR was performed to determine the effects of FN siRNA or different integrin antibodies on the mRNA expression levels of FN1 and COL1A1 in cancer-associated fibroblast cell lines CAF1299 and CAF1424 (Fig. 4). The RT-PCR results showed that antibodies targeting integrins α5β1 and αvβ3 did not reduce the mRNA expression of FN1 and COL1A1.

[0222] Western blot analysis showed that targeting integrins α5 and β3 using siRNA or antibodies reduced the expression of fibronectin substrates and induced the expression of soluble collagen I (Fig. 5). The effects of different integrin antibodies on fibronectin and collagen I in CAF1299 cells are shown in Fig. 6. Immunofluorescence staining results showed the effects of different integrin antibodies on the structure and abundance of extracellular fibronectin and collagen I. CAF1299 cancer-associated fibroblasts were cultured for 72 hours to assemble fibronectin and collagen I. Subsequently, cells were treated with different integrin antibodies (10 μg / mL) for up to 96 hours (replenishing with new antibodies every 48 hours), then fixed with 4% PFA and blocked with 1× HBSS buffer containing 5% goat serum, followed by immunofluorescence staining for fibronectin and collagen I. Antibodies targeting integrins α5β1 and αvβ3 were shown to disrupt pre-existing fibronectin and collagen I substrates (Fig. 6).

[0223] CAF1299 cancer-associated fibroblasts were cultured for 72 hours to assemble fibronectin and collagen I substrates. Subsequently, the cells were treated with different integrin antibodies (IgG4, anti-αvβ3 (ABT101), anti-α5β1 (ABT701), or anti-αvβ3 / α5β1 (ABT601), with all antibodies at a concentration of 10 μg / mL) for up to 96 hours (replenishing with new antibodies every 48 hours), followed by Picrosirius Red staining to stain total fibrous collagen. The effects of the different integrin antibodies demonstrate that antibodies targeting integrins α5β1 and αvβ3 can prevent the production and assembly of fibrous collagen (Fig. 7).

[0224] CAF1299 cancer-associated fibroblasts were cultured for 72 hours to assemble fibronectin and collagen I substrates. Subsequently, the cells were treated with different integrin antibodies (IgG4, anti-αvβ3 (ABT101), anti-α5β1 (ABT701), or anti-αvβ3 / α5β1 (ABT601), with all antibodies at a concentration of 10 μg / mL) for up to 96 hours (replenishing with new antibodies every 48 hours), followed by Picrosirius Red staining to stain total fibrous collagen. As shown in Figure 8, antibodies targeting integrins α5β1 and αvβ3 can disrupt pre-existing fibronectin and collagen I substrates. However, antibodies targeting integrin α5β1 and / or αvβ3 do not affect the attachment of fibroblasts to fibronectin or collagen I substrates (Fig. 9).

[0225] Human pancreatic ductal adenocarcinoma cells, KP4 and PANC1, were cultured at a concentration of 10 μg / mL of IgG4, the monoclonal antibodies anti-αvβ3 (ABT101) or anti-α5β1 (ABT701), or the bispecific antibody anti-αvβ3 / α5β1 (ABT601) of the example, and the cells were fixed at the final stage. FAC binding data for the three antibodies show that the binding of the bispecific anti-αvβ3 / α5β1 (ABT601) to pancreatic tumor cells in cells fixed at the final stage was enhanced at 10 μg / mL compared to the monoclonal antibodies (anti-αvβ3 and anti-α5β1 (ABT101 and ABT701), respectively) (Figure 10).

[0226] PANC1 cells were cultured with antibodies (10 μg / mL): IgG4 (control), anti-αvβ3 (ABT101), anti-α5β1 (ABT701), and anti-αvβ3 / α5β1 (ABT601), then seeded onto uncoated or fibronectin-coated plates supplemented with serum-free medium and cultured for 24 hours. The effects of different antibody treatments on the expression of YAP and its two known sub-targets, CTGF and CYR61, in pancreatic cancer cell line PANC1 cells were confirmed in Figure 11. These results demonstrate that the expression of YAP targets is not upregulated in tumor cells cultured in the presence of antibodies targeting integrins α5β1 and αvβ3 (Figure 11). In this example, YAP targets serve as a general readout indicator of the cellular response to fibronectin in terms of biological reprogramming toward a more mesenchymal and stem cell-like phenotype.

[0227] Fresh lung tumor tissue was cut into 1–2 mm thick sections using a scalpel (Day 0). The tissue was immediately collected and fixed to be used as a sample representing Day 0 (d0). For each condition, two randomly selected sections from different sites of each tumor were used. The sections were placed in a chamber of a 24-well Transwell® plate with 1 mL of complete DMEM media placed in the bottom chamber. After 24 hours, the sections were treated with antibodies at a concentration of 10 μg / mL. The medium was replaced every 48 hours with a medium containing fresh antibodies. 72 hours after treatment (Day 4, d4), the samples were fixed with formalin. The formalin-fixed, paraffin-embedded samples were stained with trichrome blue, and the staining results were analyzed using QuPath to determine the percentage of blue area. As shown in Figure 12, the bispecific antibody ABT601 reduced the collagen content in lung tissue samples more than ABT701 or ABT101, whereas ABT701 and ABT101 increased the percentage of the area occupied by collagen.

[0228] Example 3: Tumor cells overcome isolation stress by using FN generated from CAF.

[0229] FN plays a crucial role in ECM assembly and remodeling during various physiological and pathological responses. To evaluate the importance of FN generated by CAFs on tumor initiation, human PDAC cells, specifically KP4 cells, were stereotactically injected into the pancreas of immunodeficient nu / nu mice, either alone or mixed with PDAC-derived CAFs in a 1:1 ratio, and tumor initiation in the pancreas was assessed (Fig. 13A). After 3 weeks, bioluminescent imaging results showed that actual tumors (luciferase signal >1e+06) were identified in both 5 / 5 mice of the group injected with KP4 cells and CAFs, whereas no tumors were identified in both 5 / 5 mice of the group injected with KP4 cells alone (0 / 5), confirming that tumor initiation in this model is dependent on CAFs. On the other hand, CAFs in which FN expression was suppressed using siRNA were able to induce tumorigenesis (characteristics of tumor stem cells) in only 1 out of 5 animals, suggesting that FN generated by CAFs contributes significantly to the role of CAFs in this process. Considering that the efficiency of gene expression suppression using siRNA begins to decrease from day 6 after transformation, this data suggests that FN generated by CAFs is essential in the early stages of tumorigenesis in vivo.

[0230] Example 4: The interaction between CAF and fibronectin provides an essential support for collagen fiber deposition.

[0231] Previous studies have identified FN as a scaffold that coordinates the assembly and integration of additional proteins and secretory factors (Wu et al. (2023) Nat. Cell Biol. 25(2):309-322). Indeed, CAFs isolated from PDAC patients produce a fibrous ECM that clearly exhibits a high-level co-pattern of FN and collagen (COL) fibers (Fig. 13B). As evidence that FN serves as a crucial support for the assembly of additional substrate proteins, inhibition of fibronectin (FN1) expression using siRNA prevents CAFs from generating collagen fibers (Fig. 13B). The reduced degree to which collagen is assembled into insoluble fibers is consistent with the increased "soluble collagen" content in cultures collected from CAFs with FN knockdown compared to scrambled controls (Fig. 13C). In contrast, inhibition of type 1 collagen alpha 1 chain (COL1A1) expression does not affect FN fiber assembly, which highlights that FN serves as an important underlying support for the stacking of additional ECM components such as collagen. Taken together, these results not only demonstrate that FN is essential for CAF-mediated collagen fiber formation but also show that genetic approaches to disrupt this process establish proof of concept for the potential targeting of the corresponding target.

[0232] Example 5: FN-linked integrins are very important for the formation of fibrotic substrates.

[0233] Previous studies have confirmed that activated fibroblasts (myofibroblasts) utilize cell surface integrins as anchor points to apply tension to fibronectin molecules, thereby exposing potential binding sites necessary for polymerization into FN “fibers” and providing a physical scaffold and reservoir for assembling and recruiting additional oncogenic factors to support the invasive behavior of cancer cells (Liu et al. (2018) Clin. Cancer Res. 24(10):2370-2382; Kim et al. (2022) Theranostics 12(12):5258-5271; Westphalen et al. (2016) Cell Stem Cell 18(4):441-55; Chandrakesan et al. (2017) Mol. Cancer 16(1):30). Although several integrins can act as receptors for FN, integrins αvβ3 and α5β1 are not present in normal cells but are upregulated in various activated cell types, including myofibroblasts (Cooper & Giancotti (2019) Cancer Cell 35(3):347-367; Carlson et al. (2019) Nat. Cell Biol. 21(2):238-250; Knowles et al. (2013) Cancer Res. 73(20):6175-84). Indeed, CAFs isolated from human pancreatic cancer tumors exhibit potent protein expression of both α5 and β subunits (Fig. 14A) and cell surface expression of αvβ3 and α5β1 heterodimers. Similar to the inhibition of FN1 expression, collagen fiber formation can be reduced by the inhibition of the expression of the integrin β3 subunit (ITGB3, a limiting factor for αvβ3 heterodimer formation) or the integrin α5 subunit (ITGA5, which similarly regulates α5β1 expression) (Fig. 14A).Imaging of permeable cells revealed that while inhibition of FN or FN-binding integrin expression did not completely eliminate the collagen-producing ability of CAF (as evidenced by the presence of intracellular collagen), it significantly eliminated the ability of CAF to assemble extracellular collagen into fibers (Fig. 14A). Taken together, these results indicate that FN-integrin interactions on the surface of CAF are essential for the formation of FN fibers that function as scaffolds for other fibrotic matrix proteins, and further suggest that targeting integrins α5β1 and αvβ3 offers the potential to significantly inhibit CAF's contribution to cancer progression. Since integrins αvβ3 and α5β1 are generally rarely expressed in normal cells but are expressed at high levels in CAF, targeting their functions may provide an opportunity to selectively inhibit the pathological fibrosis supporting the progression of PDAC.

[0234] Example 6: Integrin blocking antibody prevents and reverses extracellular matrix (ECM) assembly by CAF

[0235] To simultaneously target two integrins required for ECM generation by CAF using a single drug, the inventors designed a novel bispecific antibody (ABT601) for dual monovalent recognition of integrin α5β1 and αvβ3 heterodimers (Fig. 2A). First, ABT601 was compared to two parental control bivalent monoclonal antibodies (mAbs) that individually recognize either integrin αvβ3 or α5β1. Commercially available antibodies for recognizing integrin αvβ3 and integrin α5 (P1D6) were included as additional reference materials. Flow cytometry was used to evaluate the extent to which each antibody (tested at a concentration of 10 μg / mL) bound to CAF-1299 and CAF-1424, CAF cell lines derived from pancreatic cancer patients. In both CAF models, the median fluorescence intensity (MFI) signal of ABT601 was slightly higher than the sum of the signals of its two parent antibodies (Figs. 15A and 15B).

[0236] Consistent with the effects of genetically repressing β3 or α5, treatment of CAF with mAbs recognizing αvβ3 or α5β1 resulted in varying degrees of reduction in the assembly of FN and COL fibrils, which may reflect the relative expression levels of these integrins in specific cell populations. Meanwhile, ABT601, which dually recognizes integrins αvβ3 and α5β1, blocked the formation of FN and COL fibrils more completely than treatment with any single monoclonal antibody alone (Fig. 16A). Immunostaining of CAF for FN and actin revealed punctate FN expression in antibody-treated cells that was no longer aligned with actin fibrils, confirming that these antibodies did not affect the substrate adhesion or cytoskeleton of the cells. Performing decellularization (i.e., CAF removal) after 72 hours of treatment leaves behind the "CAF-ECM" substrate, which clearly demonstrates the significant effect of ABT601 on both FN and COL fibers. Similar effects of ABT601 were observed in primary hepatic stellate cells isolated from patients with liver fibrosis and primary lung fibroblasts isolated from patients with fibrotic lung disease; this suggests that this antibody has the potential for widespread use as a universal antifibrotic agent targeting activated fibroblasts that rely on FN and integrins α5β1 / αvβ3 for substrate assembly. Consistent with the fact that changes occur in substrate assembly rather than substrate generation, treating CAF with ABT601 FN1 and COL1A1 It did not induce changes in the mRNA expression of any of the genes.

[0237] In the test tube ( in vitroTo directly compare the combination of ABT601 and a monospecific mAb, immunostaining for fibronectin was evaluated after treating CAF with the antibody for 72 hours. In these in vitro tests where cells are exposed to saturated concentrations of the antibody ( in vitro Under the conditions, all tested antibodies blocked FN production by CAF to the same extent compared to the IgG isomorphic control. Interestingly, αvβ3 mAb significantly reduced FN levels but did not induce the characteristic punctate FN observed with α5β1 mAb, ABT601, and the combination of α5β1 / αvβ3 mAb.

[0238] Integrins exist in a dynamic equilibrium state where binding events are continuously formed and dissociated. While competitive inhibitors based on the small molecule peptide Arg-Gly-Asp (RGD) do not easily reverse integrin-ligand binding, blocking β1 antibodies have been reported to increase the dissociation rate of integrin-FN complexes and act allosterically (Insua-Rodriguez et al. (2018)). EMBO Mol. Med. 10(10):e9003). To determine whether ABT601 could disrupt already formed substrates, CAFs were cultured for 72 hours before adding integrin-targeting antibodies to allow ECM proteins to be deposited. Indeed, over 3 days, ABT601 was able to disrupt FN and COL fibers already formed by CAFs (Fig. 16B), which suggests that ABT601’s ability to allosterically disrupt the continuous binding / dissociation (on / off) state of FN-integrin binding can exhibit a significant antifibrotic effect by shifting the equilibrium to a non-adherent state.

[0239] As a biological indicator of how PDAC cells respond to CAF, the inventors evaluated the ability of the ECM generated by CAF to upregulate the expression of connective tissue growth factor (CTGF) in tumor cells. CTGF is an extrastromal protein produced by both stromal cells and tumor cells, and is known to mediate interactions between these cells, promote fibrosis, and enhance tumor initiation in pancreatic cancer (Efthymiou et al. (2020) Front. Oncol. 10:641; Spada et al. (2021) J. Exp. Clin. Cancer Res. 40(1):102). CAF was cultured for 72 hours to allow substrate deposition, and then treated with a homozygous control or ABT601 for an additional 72 hours. Afterward, the CAF was removed to leave "CAF-ECM," and PANC1 cells were seeded onto it. After culturing for an additional 24 hours, PANC1 cell lysates were collected and analyzed by immunoblot. In this model, CAF-ECM significantly upregulated CTGF expression in PDAC cells, and this upregulation was inhibited in the presence of ABT601 (Fig. 16C). This analysis demonstrates that CTGF upregulation in tumor cells induced by the response to the ECM generated by CAF can be inhibited by blocking αvβ3 and α5β1, two major integrins that bind to FN.

[0240] Example 7: Inhibition of the function of FN-binding integrins inhibits the tumor-promoting ability of CAFs.

[0241] To evaluate the ability of αvβ3 / α5β1 ABT601 to inhibit tumor initiation in vivo, the inventors utilized a subcutaneous xenograft model in which the earliest stages of tumor initiation over time can be easily detected. Similar to the allograft model (Fig. 13A), PDAC cells were injected in quantities smaller than the optimal amount so that tumor initiation depended on the co-injection of CAF. In this model, when only a limited number of human PANC1 PDAC cells were subcutaneously injected, no proliferable tumors were observed even after 8 weeks post-injection, whereas when PANC1 cells and CAF were injected together in a 1:1 ratio, a tumor formation rate of 100% was observed (Fig. 17A). That is, proliferable tumors formed at all 12 injection sites where PANC1 cells and CAF were injected together, demonstrating that this model is valid as a biological indicator for evaluating CAF-dependent tumor initiation.

[0242] As observed in the allogeneic transplant model (Fig. 13A), the “increase” in CAF-dependent tumorigenesis in the subcutaneous region could be prevented by using CAF (si-FN) with FN1 expression suppressed via siRNA, whereas in the case of CAF (si-α5) with ITGA5 expression suppressed, tumors developed at 1 out of 11 injection sites. Since the expression suppression by siRNA is transient, these results suggest that CAF contributes to tumor initiation during the first few days after co-injection, when tumor cells utilize CAF to overcome isolation stress during the process of settling in the tumor initiation niche. At this critical stage, suppressing the expression of FN or integrin α5 in CAF alone can fully explain their ability to promote tumorigenesis.

[0243] Therefore, we investigated whether ABT601, a therapeutic agent designed for the dual targeting of integrins αvβ3 / α5β1, could exert similar activity in blocking tumor initiation. Since ABT601 recognizes antigens of most species excluding mice, its effect on tumor initiation in this xenograft model can be attributed to direct binding to antigens on human CAFs injected alongside human PDAC cells, and not to binding to integrins present on the surface of mouse stromal cells or angiocytes. Tumor cells and CAFs were pre-mixed with 10 μg / mL of ABT601 immediately before injection. After injecting the tumor cells, ABT601 was administered systemically via intraperitoneal injection at a dose of 10 mg / kg twice a week during the experiment. Notably, in mice treated with ABT601, an αvβ3 / α5β1 bispecific antibody, palpable tumors were formed at only 2 out of 12 injection sites (Fig. 17A), suggesting that the enhancement effect of tumor initiation provided by CAF can be therapeutically targeted. Since the effect exhibited by the bispecific antibody is also reproduced by the inhibition of FN or α5 expression in CAF, blocking integrin function in CAF has the ability to inhibit tumor initiation.

[0244] Example 8: ABT601 treatment reduces the fibrotic effect of human CAF injected with tumor cells

[0245] To evaluate the mechanism of action of expression inhibition and blocking strategies, all palpable tumors were collected at the end of the experiment, week 8. Analysis of H&E stained sections revealed that the two tumors formed in the ABT601-treated group had significantly less stroma and extensive necrosis compared to the untreated tumors (Fig. 17B). Previous studies have shown that PDAC tumors exhibit very high stiffness primarily due to the extensive accumulation of stroma and ECM within the tumor, and this stiffness has been shown to correlate with the progression of PDAC in mice and humans (Cooper & Giancotti (2019)). Cancer Cell 35(3):347-367; Carlson et al. (2019) Nat. Cell Biol. 21(2):238-250). Consistent with this, atomic force microscopy results in mice treated with ABT601 showed significantly lower tissue stiffness throughout the tumor (Fig. 17C).

[0246] Analysis of the tumor microenvironment also supports the hypothesis that co-injection with CAF creates a highly fibrotic and reactive tumor environment that can serve as a therapeutic target. When tumors formed by co-injection of PANC1+CAF were stained using an antibody that recognizes human FN (hFN) but not mouse FN, regions with strongly stained hFN fibrous structures were observed (Fig. 18A). This suggests that co-injected CAF is the primary source of tumor stroma compared to host (i.e., mouse) fibroblasts in this model. In the tumor regions with deep hFN staining, evaluation using Mason tristaining (Fig. 18B) and Picrosirius Red staining for collagen fibers (Fig. 18C) revealed significant fibrosis (collagen polymerization). In contrast, in tumors of mice treated with ABT601, hFN was completely eliminated, while the number and size of collagen fibers were reduced. Since ABT601 used in this study does not recognize mouse antigens, it can only identify injected human PDAC cells and human CAFs in this xenograft model. This specificity suggests that the interaction between co-injected PANC1 cells and CAFs mediates the earliest stages of tumorigenesis prior to the influx of mouse stromal cells into the tumor microenvironment. Analysis of serial sections of tumors stained with hFN and collagen suggests that human-derived CAFs contribute to the generation of highly fibrotic stroma, whereas mouse host cells that do not respond to ABT601 are likely the source of less developed collagen networks.

[0247] In addition to the observed inhibition of FN generated by CAF, the inventors predict that ABT601 in humans may obtain additional antitumor efficacy by acting on activated cell types within tumors known to exhibit increased αvβ3 / α5β1 expression, including angiogenic endothelial cells and tumor-associated macrophages. In a xenograft model, a decrease in immunohistochemical intensity for CD31, an endothelial cell marker used to evaluate tumor angiogenesis, was observed. Therefore, ABT601 has the potential to exert an indirect anti-angiogenic effect on the mouse vascular system by inhibiting the accumulation of stimulating factors and ECM proteins that induce vascular cells to generate new blood vessels within the tumor microenvironment (Fig. 18D) (Knowles et al. (2013) Cancer Res. 73(20):6175-84; Nagpal et al. (2023) Cancers (Basel) 15(4):1216).

[0248] This study revealed that when PDAC cells are exposed to “isolation stress” occurring at various stages of tumor development and progression, the assembly of FN and COL fibers mediated by CAF is particularly promoted and enhanced. The inventors demonstrate that inhibiting the ability of CAF to organize FN fibers halts COL fibrin formation, thereby disrupting intercellular communication and providing an opportunity to suppress tumor cell survival for a sufficient amount of time to “form” new tumor clusters (Fig. 19).

[0249] Example 9: ABT601 reduces fibrotic response in metabolic dysfunction-associated steatohepatitis (MASH) model

[0250] To evaluate the effect of ABT601 on the fibrotic response induced by metabolic dysfunction-associated steatohepatitis (MASH), a human liver spheroid model (Kim et al. (2024) STAR Protoc. 5(2):103111) was used. Primary hepatocytes isolated from human donors were mixed with ABT601 and cultured for 7 days to form spheroids, after which they were stimulated with a MASH cocktail. On day 14, spheroids and supernatants were collected and analyzed (Fig. 20A). Brightfield images were taken on day 14, and the diameter of the spheroids was measured and normalized using ImageJ software (Fig. 20B). ELISA was performed to measure collagen I in the spheroid supernatant (Fig. 20C), and qPCR was performed on the spheroid lysate to analyze genes important for the fibrotic response (Fig. 20D). These analysis results indicated that ABT601 reduced the fibrotic response in the MASH model.

[0251] order:

[0252]

[0253]

Claims

Claim 1 A bispecific antibody or its antigen-binding fragment that specifically binds to two different integrins selected from integrin αvβ3 and integrin α5β1. Claim 2 A bispecific antibody or an antigen-binding fragment thereof, wherein, in claim 1, it comprises a first variable domain that specifically binds to integrin αvβ3 and / or a second variable domain that specifically binds to integrin α5β1, wherein the first variable domain and the second variable domain are different. Claim 3 A bispecific antibody or an antigen-binding fragment thereof, wherein the first variable domain specifically binds to integrin αvβ3 and the second variable domain specifically binds to integrin α5β1. Claim 4 A bispecific antibody or an antigen-binding fragment thereof, wherein, in any one of claims 1 to 3, the Fc domain of an IgG antibody is further included. Claim 5 In paragraph 4, the Fc domain is a bispecific antibody or an antigen-binding fragment thereof, wherein the Fc domain is the Fc domain of an IgG1 antibody or an IgG4 antibody. Claim 6 In any one of paragraphs 3 through 5, the first domain is Sequence No. 1 A heavy chain VH-(AS)-hk.LC.h4.HC.S228P.T366W comprising the amino acid sequence of or a sequence having at least 90% identity with the same, and Sequence No. 2 A bispecific antibody or its antigen-binding fragment comprising a light chain VL-(SS)-h1v2.CH1 having an amino acid sequence or at least 90% identity therewith. Claim 7 In any one of paragraphs 3 through 6, the second domain is Sequence No. 3 A heavy chain VH-(AS)-h4.HC.S228P.T363S.V366A.Y404V comprising the amino acid sequence of or a sequence having at least 90% identity with the same, and Sequence No. 4 A bispecific antibody or its antigen-binding fragment comprising a light chain VL-hk.LC having an amino acid sequence or at least 90% identity therewith. Claim 8 A bispecific antibody or its antigen-binding fragment, wherein, in any one of claims 1 to 7, the first and / or second variable domain is a humanized or human antibody domain. Claim 9 A polynucleotide encoding a bispecific antibody of any one of claims 1 to 8 or an antigen-binding fragment thereof. Claim 10 A vector containing the polynucleotide of claim 9. Claim 11 A host cell comprising the polynucleotide of claim 9 or the vector of claim 10. Claim 12 A composition comprising a bispecific antibody of any one of claims 1 to 8, an antigen-binding fragment thereof, and a carrier. Claim 13 A pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. Claim 14 A pharmaceutical composition comprising additional therapeutic agents in Clause 13. Claim 15 In paragraph 14, the additional therapeutic agent is a pharmaceutical composition that is a chemotherapy agent. Claim 16 A kit comprising a bispecific antibody of any one of claims 1 to 8 or an antigen-binding fragment thereof. Claim 17 A method for preventing a cell from binding to fibronectin, comprising the step of contacting a cell with an effective amount of an antibody that specifically binds to one or more integrins or an antigen-binding fragment thereof. Claim 18 In paragraph 17, the antibody is anti-α5β1 (ABT701), method. Claim 19 A method according to claim 17, wherein the antibody or its antigen-binding fragment is the bispecific antibody or its antigen-binding fragment of any one of claims 1 to 8. Claim 20 In paragraph 19, a method wherein a bispecific antibody or an antigen-binding fragment thereof prevents a cell from binding to fibrillar fibronectin. Claim 21 In paragraph 19, a method wherein a bispecific antibody or its antigen-binding fragment prevents a cell from binding to spherical fibronectin. Claim 22 A method according to any one of claims 19 to 21, wherein the bispecific antibody or its antigen-binding fragment prevents the cell from binding to both fibrous and globular fibronectin. Claim 23 A method according to any one of paragraphs 17 to 22, wherein the cell is a fibroblast or a mesenchymal cell. Claim 24 A method according to any one of claims 17 to 23, which prevents collagen organization by preventing fibronectin from binding to cells. Claim 25 A method for treating cancer expressing integrin αvβ3 and / or integrin α5β1 in a subject requiring treatment, comprising treating the cancer by administering to the subject an antibody that specifically binds to one or more integrins or an antigen-binding fragment thereof in a therapeutically effective amount. Claim 26 In paragraph 25, the antibody is anti-α5β1 (ABT701), method. Claim 27 A method according to claim 25, wherein the antibody or its antigen-binding fragment is a bispecific antibody or its antigen-binding fragment of any one of claims 1 to 8, or a pharmaceutical composition of any one of claims 13 to 15, thereby treating cancer. Claim 28 A method for treating a fibrotic disease comprising cells expressing integrin αvβ3 and / or integrin α5β1 in a subject requiring treatment, comprising treating the fibrotic disease by administering to the subject an antibody that specifically binds to one or more integrins or an antigen-binding fragment thereof in a therapeutically effective amount. Claim 29 In paragraph 28, the antibody is anti-α5β1 (ABT701), method. Claim 30 A method according to claim 28, wherein the antibody or its antigen-binding fragment is a bispecific antibody or its antigen-binding fragment of any one of claims 1 to 8, or a pharmaceutical composition of any one of claims 13 to 15, thereby treating a fibrotic disease. Claim 31 A method according to any one of paragraphs 28 to 30, wherein the fibrotic disease is ocular fibrosis, cardiac fibrosis, hepatic fibrosis, intestinal fibrosis, pulmonary fibrosis, pancreatic fibrosis, renal fibrosis, non-alcoholic fatty liver disease, systemic sclerosis, idiopathic pulmonary fibrosis, fibromyalgia, keloid, or cutaneous fibrosis. Claim 32 A method according to any one of claims 28 to 31, wherein the antibody or its antigen-binding fragment, or the pharmaceutical composition, is administered intravenously, subcutaneously, or intramuscularly to a subject. Claim 33 A method according to any one of claims 28 to 32, wherein the cell comprises fibroblasts and / or mesenchymal cells. Claim 34 A method for treating cancer in a subject requiring cancer treatment, comprising the following steps: a) selecting a subject having cancer cells rich in integrin αvβ3 and / or integrin α5β1, and selectively immune effector cells (e.g., macrophages); and b) treating the cancer by administering to the subject a therapeutically effective amount of an antibody that specifically binds to one or more integrins or an antigen-binding fragment thereof. Claim 35 In paragraph 34, the antibody is anti-α5β1 (ABT701), method. Claim 36 A method according to claim 34, wherein the antibody or its antigen-binding fragment is a bispecific antibody or its antigen-binding fragment of any one of claims 1 to 8, or a pharmaceutical composition of any one of claims 13 to 15, thereby treating cancer. Claim 37 A method according to claim 34, wherein step a) comprises obtaining a sample of cancer from a subject and measuring the levels of integrin αvβ3 and / or integrin α5β1 and optionally the levels of immune effector cells in the sample. Claim 38 A method according to any one of paragraphs 34 to 37, wherein the cancer is epithelial carcinoma, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, kidney cancer, breast cancer, brain cancer (e.g., glioma, e.g., glioblastoma multiforme), skin cancer, stomach cancer, prostate cancer, esophageal cancer, or colorectal cancer. Claim 39 A method according to any one of paragraphs 34 to 38, wherein the cancer comprises a KRAS mutation or an EGFR mutation. Claim 40 A method according to any one of paragraphs 34 through 39, wherein the tumor of the cancer is rich in macrophages. Claim 41 A method according to any one of claims 34 to 40, wherein the cancer is a cellular carcinoma, optionally wherein the cellular carcinoma is a terminal cellular carcinoma, and optionally wherein one or more epithelial cells of the cancer have been at least partially converted into mesenchymal cells. Claim 42 In paragraph 41, the method wherein the epithelial cell carcinoma is resistant or refractory to chemotherapy, or has become resistant or refractory to chemotherapy. Claim 43 A method comprising, in any one of paragraphs 34 to 42, further comprising the step of administering an additional cancer treatment or treatment to a subject. Claim 44 In any one of paragraphs 34 to 43, the antibody or its antigen-binding fragment or pharmaceutical composition is administered intravenously, subcutaneously, or intramuscularly to a subject, or is in situ (in the tumor or around the tumor) in situ A method of being injected into ). Claim 45 A method in any one of paragraphs 25 through 44, wherein the subject is a human. Claim 46 A method for inhibiting collagen organization in a subject, comprising the step of administering a composition targeting fibronectin to a subject. Claim 47 In paragraph 46, the composition targets one or more fibronectin receptors. Claim 48 In paragraph 47, the composition targets two or more fibronectin receptors. Claim 49 A method according to claim 47 or 48, wherein the composition inhibits the expression or action of one or more fibronectin receptors. Claim 50 A method according to any one of claims 47 to 49, wherein the fibrotic cells comprise one or more fibronectin receptors. Claim 51 A method according to any one of claims 46 to 50, wherein the composition comprises an antisense oligonucleotide, RNAi, or siRNA. Claim 52 A method according to any one of claims 46 to 50, wherein the composition comprises an antibody that specifically binds to fibronectin or an antigen-binding fragment thereof. Claim 53 In paragraph 52, the antibody or its antigen-binding fragment specifically binds to one or more integrins. Claim 54 A method according to claim 53, wherein the antibody or its antigen-binding fragment is the bispecific antibody or its antigen-binding fragment of any one of claims 1 to 8. Claim 55 In paragraph 46, the composition is the polynucleotide of paragraph 9, method. Claim 56 A method for reversing collagen organization in a subject, comprising the step of administering a composition targeting fibronectin. Claim 57 In paragraph 56, the composition targets one or more fibronectin receptors, a method. Claim 58 In paragraph 57, the composition targets two or more fibronectin receptors, a method. Claim 59 The method of claim 57 or 58, wherein the composition inhibits the expression and / or action of one or more fibronectin receptors. Claim 60 A method according to any one of claims 56 to 59, wherein the composition comprises an antisense oligonucleotide, RNAi, or siRNA. Claim 61 A method according to any one of claims 56 to 59, wherein the composition comprises an antibody that specifically binds to fibronectin or an antigen-binding fragment thereof. Claim 62 In paragraph 61, the antibody or its antigen-binding fragment specifically binds to one or more integrins. Claim 63 A method according to claim 62, wherein the antibody or its antigen-binding fragment is the bispecific antibody or its antigen-binding fragment of any one of claims 1 to 8. Claim 64 In paragraph 62, the composition is the polynucleotide of paragraph 9, method. Claim 65 A method for inhibiting Yes-associated protein 1 (YAP) or a YAP target gene in a subject, comprising the step of administering an antibody that specifically binds to one or more integrins or an antigen-binding fragment thereof. Claim 66 In paragraph 65, the antibody is anti-α5β1 (ABT701), method. Claim 67 A method according to claim 65, wherein the antibody or its antigen-binding fragment is the bispecific antibody of any one of claims 1 to 8, or its antigen-binding fragment, the polynucleotide of claim 9, or the vector of claim 10. Claim 68 In paragraph 65, the method wherein the YAP target gene is CTGF or CYR61.

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