Compositions and methods for treating cancer and fibrotic disorders

Abstract

The present invention relates to antibodies (e.g., bispecific antibodies, or antigen-binding fragments thereof), and compositions comprising the same. The invention further relates to polynucleotides encoding the antibodies, or antigen-binding fragments thereof, and vectors and host cells comprising the same. The invention further relates to methods of using the antibodies, or antigen-binding fragments thereof, to prevent binding of the cell to fibronectin and methods of treating cancers and fibrotic disorders. The invention further relates to the simultaneous inhibition of ligand binding to a specific combination of integrin receptors. The invention further relates to the inhibition of expression of a predetermined combination of integrin receptor subunits through nucleic acid action on the mRNAs.

Description

COMPOSITIONS AND METHODS FOR TREATING CANCER AND FIBROTIC DISORDERSSTATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 619,144, filed January 9, 2024, the entire contents of which are incorporated by reference herein.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers CA220512, T32IR4672, T29FT0343 and K010D030513 awarded by the National Institutes of Health. The government has certain rights in the invention.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0003] A Sequence Listing in XML format, entitled 1548-7WO_ST26.xml, 17,519 bytes in size, generated on December 24, 2024, and filed herewith, is hereby incorporated by reference in its entirety for its disclosures.FIELD OF THE INVENTION

[0004] The present invention relates to antibodies (e.g., bispecific antibodies), or antigenbinding fragments thereof, and compositions comprising the same. The invention further relates to polynucleotides encoding the antibodies, or antigen-binding fragments thereof, and vectors comprising the same. The invention further relates to methods of using the antibodies, or antigen-binding fragments thereof to prevent binding of a cell to fibronectin and methods of treating fibrotic disorders and cancers. The invention further relates to the simultaneous inhibition of ligand binding to a specific combination of integrin receptors. The invention further relates to the inhibition of expression of a predetermined combination of integrin receptor subunits through nucleic acid action on the mRNAs.BACKGROUND OF THE INVENTION

[0005] Fibrosis, or scarring, arises from the excess and dysregulated accumulation of extracellular matrix (ECM) components. Strategies to limit fibrosis could have applications for a wide variety of diseases. If highly progressive, fibrosis can eventually lead to organ malfunction and death. Fibrosis is a pathological feature of most chronic inflammatorydiseases. Fibrosis is also a critical component of the tumor microenvironment that supports tumor progression and enables metastatic spread.

[0006] One key mediator of fibrosis is fibronectin, a large glycoprotein that can influence a number of important cellular functions. It plays a major role in wound healing and embryonic development, yet is also associated with a number of pathologies including cancer and fibrosis. Fibronectin acts as a provisional matrix protein that functions as a scaffold for depositing other ECM components, such as collagens, proteoglycans, and growth factors, ultimately regulating ECM elaboration and architecture. For example, fibronectin is indispensable for type I collagen polymerization and fibril assembly 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) and cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin (Erdogan et al. (2017) J. CellBiol. 216:3799-3816). Thus, interfering with fibronectin function may provide an opportunity to destabilize or disempower the fibronectin scaffold that is critical for the formation of additional ECM components that contribute to the progression of a variety of diseases including cancer.

[0007] Cells interact with fibronectin using specialized cell surface receptors, such as integrins and proteoglycans such as syndecans. Integrins are expressed in most cells of multicellular animals, with most adult mammals constitutively expressing multiple different integrins. Integrins have large extracellular domains for ligand binding; multiple alpha and beta subunits have been identified, with these subunits combining to form distinct integrin heterodimers. Integrins are comprised of a and P subunits that form a family of at least 24 different heterodimeric cell surface receptors that can have overlapping or unique physiological functions. Integrins a5pi and avP3 primary receptors that cells use to bind fibronectin, although some additional integrins can also participate in some cell types, including avpi, avP6, avP8, a4pi, a4p7, and a9pi.

[0008] The progression of many pathologies including fibrotic diseases and cancer are promoted when various cell types produce and / or respond to fibronectin. Fibroblasts are stromal cells that, when activated, produce and secrete fibronectin molecules into their pericellular environment. Integrins on the surface of fibroblasts bind and anchor these “globular” fibronectin molecules to allow larger fibronectin “fibrillar” polymers to be formed. Fibrillar fibronectin acts as a scaffold to support and anchor additional extracellular matrix proteins such as collagen and proteoglycans. If not tightly regulated, fibronectin production can trigger the development of a fibrotic environment with properties such as increased matrix stiffness and sequestration of activating factors that promotes and exacerbates a variety ofdisease states such as fibrotic disease and cancer. Preventing fibroblasts from utilizing integrins to interact with fibronectin provides an opportunity to suppress the formation of fibrillar fibronectin polymers and thus halt this sequence of events that leads to fibrosis.

[0009] Besides fibroblasts, other cell types can utilize integrins to interact with fibronectin and its associated extracellular matrix proteins. For example, preventing tumor cells from interacting with fibronectin may prevent their reprogramming toward a more aggressive and stemlike state. Similarly, preventing endothelial cells from engaging fibronectin may suppress their proliferation and invasion that mediate pathological angiogenesis in a variety of settings. Thus, an agent that can disrupt fibronectin receptors from engaging fibronectin has the potential to provide therapeutic value for a variety of diseases including cancer and fibrosis.

[0010] Antibodies are proteins that bind to a specific antigen. Antibody binding to an antigen selectively expressed on a cell may produce anti-fibrotic or anti-tumor effects by directly blocking the function of the antigen that promotes tumor cell growth or survival pathways. As detailed above, preventing a variety of different cell types from interacting with fibronectin could provide therapeutic benefit. In the setting of cancer, an antibody can also act as a bridge to bring together a tumor cell with an immune effector cell that can indirectly induce tumor cell destruction. This method of “antibody dependent” tumor cell killing mediated by immune effector cells might work additively or synergistically with its direct function blocking activity to prevent tumor cell biological reprogramming by preventing a cell’s interaction with fibronectin. In this context, antibodies that target fibronectin-binding integrins may have dual and complementary mechanisms of action that include activity on multiple cell types that are implicated in the initiation and progression of cancer.

[0011] Recently, a bispecific antibody targeting all av integrins in addition to a5pi has been developed with improved properties relative to a monospecific antibody in prostate cancer cells (Joshi et al. (2020) Mol. Cancer Res. 18(l):27-32). Additional bispecific antibodies have been described (Liu et al. (2015) Mol. Pharm. 12(7):2544-50; Shlamkovich et al. (2018) BMC Biol. 16:92; Gallo et al. (2021) J. Mol. Biol. 443: 15; International Patent Publication WO 2019 / 089544 Al; Joshi et al. (2019) Cancer Res. 79(13_Suppl.):902; Bhatta et al. (2021) MAbs. 13(1): 1859049). However, there remains a need for new compositions, and methods of using such compositions, for example, to treat fibrotic diseases and prevent cellular interaction with fibronectin.SUMMARY OF THE DISCLOSURE

[0012] The present invention is based not only on the understanding of how fibroblasts utilize integrins to create a fibrillar fibronectin scaffold that supports generation of a more elaborate extracellular matrix, but also on an understanding of how various cell types benefit from interaction with fibronectin. Specific targeting of these interactions is critical, as an antibody that simply recognizes an integrin subunit (such as alpha-v) that is a part of one possible fibronectin receptor could aberrantly disrupt the function of many normal cells containing the integrin.

[0013] Because formation of integrin heterodimers depends on the presence of both subunits, heterodimers a5pi, avP3, and avP6, each of which bind fibronectin, have more limited expression in normal, non-activated epithelium. Thus, such integrin heterodimers are more desirable cell antigens for targeted treatments based on their low expression in normal adult tissues and their selective enrichment on epithelial tumors as they become more aggressive, late-stage, and drug resistant.

[0014] Another function of a therapeutic antibody is to engage effector cells for tumor killing or to be conjugated to a drug to induce tumor cell killing. Utilizing an antibody that targets the mesenchymal-like integrins such as P3, a5, and P6 that are co-enriched with macrophages provides the possibility of optimal tumor-selective killing. Because certain tumors may require different effector cells, a system that allows for flexibility in the integrin antigens with various modes of antibody-mediated killing would be advantageous. Examples include engagement of immune effector cells to effectively mediate antibody-dependent cellular cytotoxicity (ADCC) against epithelial cancer cells and / or antibody engagement of macrophages as an effector cell to promote antibody-dependent cellular phagocytosis (ADCP).

[0015] Without being bound by theory, preventing fibronectin binding to more than one integrin using a single therapeutic agent may provide better therapeutic coverage across a heterogeneous population when the antigens are recognized on different tumor cells, may provide better recognition, killing and / or inhibition when both antigens are recognized on a single tumor cell, enhancing homing to the tumor more effectively, and / or allow for inhibition of binding to both globular and fibrillar forms of fibronectin. In an embodiment, inhibiting cellular engagement of fibronectin by simultaneously disrupting fibronectin interactions with two integrins, e.g., a5p l and av 3, can provide a more effective blocking of binding, thereby inhibiting further cancer or fibrotic disorder progression,

[0016] Thus, one aspect of the invention relates to a bispecific antibody, or antigen-binding fragment thereof, that binds two different integrins, a5pi and avp3.

[0017] An aspect of the invention relates to a bispecific antibody or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi, and compositions or pharmaceutical compositions comprising the bispecific antibody or antigen-binding fragment thereof.

[0018] Another aspect of the invention relates to a polynucleotide encoding the bispecific antibody or antigen-binding fragment thereof of the invention and vectors and host cells comprising the polynucleotide.

[0019] An additional aspect of the invention relates to methods of inhibiting the binding of a cell to fibronectin, comprising contacting the cell with an effective amount of an antibody or antigen-binding fragment thereof that targets one or more fibronectin-binding integrin (e.g., a bispecific antibody, or antigen-binding fragment thereof).

[0020] Another aspect of the invention relates to method of treating a cancer expressing integrin avP3 and integrin a5pi in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof that targets one or more fibronectin-binding integrin (e.g., a bispecific antibody, or antigen-binding fragment thereof), or the pharmaceutical composition of the invention to the subject, thereby treating the cancer.

[0021] A further aspect of the invention relates to a method of treating an epithelial cell cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof that targets one or more fibronectin-binding integrin (e.g., a bispecific antibody or antigen-binding fragment thereof) or the pharmaceutical composition of the invention to the subject, thereby treating the epithelial cell cancer.

[0022] Another aspect of the invention relates to a method of treating a cancer in a subject in need thereof, comprising the steps of: a) selecting a subj ect having cancer cells that are enriched for integrin avP3 and / or integrin a5pi, and optionally an immune effector cell (e.g., a macrophage); and b) administering a therapeutically effective amount of an antibody or antigenbinding fragment thereof that targets one or more fibronectin-binding integrin (e.g., a bispecific antibody or antigen-binding fragment thereof) or the pharmaceutical composition of the invention to the subject, thereby treating the cancer.

[0023] Another aspect of this invention relates to a method of treating a fibrotic disorder expressing integrin avP3 and / or integrin a5pi in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigenbinding fragment thereof that targets one or more fibronectin-binding integrin (e.g., a bispecificantibody, or antigen-binding fragment thereof), or the pharmaceutical composition of the invention to thereby treat the fibrotic disorder.

[0024] Another aspect of the invention relates to a method of inhibiting collagen organization in a subject, comprising administering a composition targeting fibronectin.

[0025] Another aspect of the invention relates to a method of disrupting existing collagen organization in a subject, comprising administering a composition targeting fibronectin.

[0026] Another aspect of the invention relates to the delivery of an inhibitory nucleic acid (e.g., siRNA), e.g., in the form of a nucleic acid, gene vector construct, or recombinant viral vector to inhibit expression of specific integrin receptor subunits. The inhibition of integrin receptor expression could be either any single integrin receptor or combination comprising integrin avP3 and / or integrin a5pi.

[0027] Another aspect of this invention relates to a method of inhibiting Yes-associated protein 1 (YAP) or YAP target genes in a subject, comprising administering an antibody or antigenbinding fragment thereof that targets one or more fibronectin-binding integrin (e.g., a bispecific antibody, or antigen-binding fragment thereof, of the present invention, a polynucleotide encoding the antibody, or antigen-binding fragment thereof, or a vector comprising the polynucleotide, or an inhibitory nucleic acid of the present invention to the subject.

[0028] A further aspect of the invention provides a bispecific antibody, or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi, in the preparation of a medicament for use in the treatment of a cancer expressing integrin avP3 and integrin a5pi in a subject.

[0029] Another aspect provides a bispecific antibody, or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi, in the preparation of a medicament for use in the treatment of epithelial cell cancer in a subject.

[0030] Another aspect provides a bispecific antibody, or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi, in the preparation of a medicament for use in the treatment of a fibrotic disorder expressing integrin avP3 and / or integrin a5pi in a subject.

[0031] Another aspect provides a bispecific antibody, or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi, in the preparation of a medicament for use toinhibit the binding of a cell to fibronectin, inhibit collagen organization, reverse collagen organization, and / or inhibit YAP or YAP target genes in a subject.

[0032] A further aspect of invention relates to a bispecific antibody, or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi, for use in the treatment of a cancer expressing integrin avP3 and integrin a5pi in a subject.

[0033] Another aspect provides a bispecific antibody, or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi, for use in the treatment of epithelial cell cancer in a subject.

[0034] Another aspect provides a bispecific antibody, or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi, for use in the treatment of a fibrotic disorder expressing integrin avP3 and / or integrin a5pi in a subject.

[0035] Another aspect provides a bispecific antibody, or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi, for use in inhibiting the binding of a cell to fibronectin, inhibiting collagen organization, reversing collagen organization, and / or inhibiting YAP or YAP target genes in a subject.

[0036] These and other aspects of the invention are set forth in more detail in the description of the invention below.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGS. 1A-1B Fibroblasts require integrins a5 and P3 for fibronectin and collagen I matrix assembly. FIG. 1A Images of CAF1299 cancer-associated fibroblast cells treated with siRNAs for 72 hours and immunostained for fibronectin and collagen I. The images for collagen I staining on the right panel show the magnified area of the box as indicated. FIG. IB. Western blotting showing the effects of different siRNAs or integrin antibodies on the protein levels of FN, collagen I, a5, and P3 in CAF1299.

[0038] FIGS. 2A-2C. Antibodies targeting integrins a5pi and avP3 significantly disrupt fibronectin and collagen I matrix assembly (shown in permeabilized fibroblasts). FIG. 2A. Bispecific antibody (ABT601) for dual recognition of avP3 and a5pi heterodimers. Schematic depicts design of novel bispecific antibody with monovalent recognition of two antigens, integrin avP3 and integrin a5pi. The Fab domains for the bispecific antibody are identical tothe “control” bivalent (z.e., monospecific) monoclonal antibodies recognizing avP3 and a5pi. FIG. 2B. Images of CAF1299 cells treated with different antibodies (10 pg / mL) for 72 hours, immunostained for fibronectin and collagen I, and imaged using confocal microscopy. The images for collagen I immunostaining on the right panel show the magnified area of the box as indicated. FIG. 2C. Western blotting showing the effects of different integrin antibodies on the protein levels of FN, collagen I, and GAPDH in CAF1299.

[0039] FIG. 3. Antibodies targeting integrins a5p 1 and avP3 significantly disrupt fibronectin and collagen matrix assembly (shown in non-permeabilized fibroblasts). CAF1299 cancer- associated fibroblast cells were treated with different antibodies (10 pg / mL) for 72 hours, then fixed with 4% PFA and blocked with IX HBSS buffer containing 5% goat serum prior to immunofluorescence staining for collagen I and fibronectin and imaging using confocal microscopy.

[0040] FIG. 4. Antibodies targeting integrins a5pi and avP3 do not reduce mRNA expression of FN1 and COL1A1. Quantitative RT-PCR showing the effects of FN siRNA or different integrin antibodies on the mRNA level of FN1 and COL1A1 in cancer-associated fibroblast cell lines, CAF1299 and CAF1424.

[0041] FIGS. 5A-5B. Targeting integrins a5 and P3 using siRNA or antibodies increases fibroblast secretion of soluble collagen I that is not incorporated into the extracellular matrix. FIG. 5A. Western blotting showing the effects of different siRNAs or integrin antibodies on the protein levels of FN, collagen I, a5, and P3 in cancer-associated fibroblast cell line, CAF1299. FIG. 5B. Western blotting showing the effects of FN siRNA or integrin antibodies on the protein level of soluble collagen I secreted by CAF1299 into the supernatant.

[0042] FIG. 6 Antibodies targeting a5pi and avP3 can disrupt pre-existing fibronectin and collagen I matrices produced by fibroblasts. Cancer-associated fibroblast cells, CAF1299, were grown for 72 hours to allow them to assemble fibronectin and collagen I. Subsequently, cells were treated with different integrin antibodies (10 pg / mL) up to 96 hours (fresh antibodies were supplemented every 48 hours), then fixed with 4% PFA and blocked with IX HBSS buffer containing 5% goat serum prior to immunofluorescence staining for fibronectin and collagen I and imaging using confocal microscopy.

[0043] FIG. 7. Antibodies targeting a5pi and avP3 can prevent fibroblast production of collagen fibers. Cancer-associated fibroblast cells, CAF1299, were grown for 72 hours to allow them to assemble fibronectin and collagen I. Subsequently, cells were treated with different antibodies (IgG4, anti-avP3 (ABT101), anti-a5pi (ABT701), or anti-avP3 / a5pl (ABT601), 10 pg / mL for all antibodies)) up to 96 hours (fresh antibodies were supplemented every 48 hours),followed by the picrosirius red staining that detects fibrillar collagen. Representative brightfield images (X8) from three random fields of each treatment are shown.

[0044] FIG. 8 Antibodies targeting a5pi and avP3 can disrupt pre-existing fibronectin and collagen I matrices produced by fibroblasts. CAF1299 cancer-associated fibroblast cells were grown for 72 hours to allow them to assemble fibronectin and collagen I. Subsequently, cells were removed and the cell-free ECM was treated with different integrin antibodies (10 pg / mL) up to 96 hours (fresh antibodies were supplemented every 48 hours), then fixed with 4% PFA and blocked with IX HBSS buffer containing 5% goat serum prior to immunofluorescence staining for fibronectin and collagen I.

[0045] FIG. 9. Antibodies targeting a5pi and / or avP3 do not affect fibroblast adhesion to fibronectin or collagen I matrices. CAF1299 cancer-associated fibroblast cells were mixed with different integrin antibodies as indicated (10 pg / mL for each) and then seeded on plasma fibronectin or collagen I-coated untreated 96-well cell culture plate. Cells were allowed to attach to the plate for up to 24 hours. Crystal violet was used to stain the adherent cells, and the absorbance at 560 nm was read by a plate reader.

[0046] FIG. 10 A bispecific antibody shows enhanced binding to the surface of pancreatic tumor cells compared to each monospecific antibody. Fluorescence Activated Cell Sorting (FACS) for human KP4 and PANCI pancreatic ductal carcinoma cells incubated with IgG4, anti-avP3 (ABT101), anti-avP3 / a5pi (ABT601) or anti-aS i (ABT701) at 10 pg / ml, cells fixed at final stage; left panels, median fluorescence intensity (MFI); right panels, histograms for each cell treatment.

[0047] FIG. 11 Yes-associated protein 1 (YAP) targets are not upregulated in tumor cells plated atop cell-free extracellular matrix produced by cancer-associated fibroblasts grown in the presence of antibodies targeting a5pi and avP3. Western blotting showing the protein levels of Yes-associated protein 1 (YAP) and two YAP targets (CTGF and CYR61) in KP4 or PANCI human pancreatic cancer cells plated atop cell-free ECM deposited by CAF cells that were treated with different integrin antibodies (10 pg / mL) for 24 hours. All cells were grown in serum-free media.

[0048] FIG. 12. Treating human lung tumor biopsy material ex vivo with a bi-specific antibody decreased collagen content. Lung tumor tissue sections showing % area of collagen after treatment with ABT701 or ABT601 antibody at day 4. Analysis with QuPath.

[0049] FIGS. 13A-13C. Tumor cells utilize CAF-produced fibronectin to overcome isolation stress. FIG. 13A. CAFs enhance tumor initiation via fibronectin (orthotopic pancreas cancer model). Luciferase-expressing KP4 PDAC cells were orthotopically injected (with or withoutan equal number of CAF-1299 cancer-associated fibroblast cells) to the pancreas of nu / nu mice. After 3 weeks, luciferase imaging was performed to survey tumor establishment in the pancreas. Table shows tumor take rate for 5 mice per group. FIG. 13B. Fibronectin knockdown prevents CAF assembly of collagen fibers. CAF-1299 and CAF-1424 cells were treated with siRNA for a scramble control vs. FN. After 72 hours, immunofluorescence staining shows FN and COL content. Images are representative of at least 3 independent experiments. FIG. 13C. Knockdown of fibronectin in CAFs increases the amount of soluble collagen secreted into the media. CAF-1299 cells were cultured for 72 hours, then the conditioned media and cell lysates were collected and processed for immunoblot to confirm fibronectin knockdown and assess the level of soluble collagen secreted by the cells into the media.

[0050] FIG. 14 Fibronectin-binding integrins mediate ECM production. Knockdown of FN or FN-binding integrins in CAFs prevents the formation of FN and COL fibers. CAF-1299 were treated with siRNA for a scramble control vs. FN, ITGA5, or ITGB3. After 72 hours, immunofluorescence staining shows FN and COL content. Images are representative of at least three independent experiments. Blots confirm knockdown.

[0051] FIGS. 15A-15B. ABT601 improves targeting of FN-binding integrins on CAFs. FIG. 15A. ABT601 binding to cells compared to commercial and control antibodies. Flow cytometry plots show binding of each antibody to CAF-1299 and CAF-1424. FIG. 15B. ABT601 shows improved binding to CAFs compared with constituent mAbs. Graph shows median fluorescence intensity (MFI) for antibody binding to CAF-1299 and CAF-1424 cells. Error bars represent standard deviation from three independent experiments.

[0052] FIGS. 16A-16C. Dual blockade of integrins avP3 / a5pi can prevent and reverse CAF- ECM assembly. FIG. 16A. Function blocking antibodies targeting FN receptors prevent CAF assembly of FN / COL fibers. CAF-1299 were incubated with control IgG vs. indicated antibodies for 72 hours, and then processed for immunostaining to examine FN and COL. Images are representative of at least three independent experiments. Graph shows the quantification of staining as mean+ / -SD for each marker that was measured as % area for each experiment, then normalized to IgG control. *P<0.05 using one-sample t-test. FIG. 16B. Integrin-targeted antibody can disrupt pre-existing CAF-produced ECM. CAF-1299 were plated and allowed to produce ECM for 72 hours before adding control IgG vs. indicated antibodies for an additional 72 hours. Samples were then processed for immunostaining to examine FN and COL. Graph shows the mean ± SD staining for each marker measured as % area in each experiment and normalized to IgG control. *P<0.05 using one-sample t-test. FIG. 16C. ABT601 prevents activation of YAP, a mechanosensor that promotes sternness. CAF-1299 were plated and allowed to produce ECM for 72 hours then treated with antibody for another 72 hours before cells were removed to leave behind a cell-free ECM atop which PANCI cells were then plated. After 24 hours, the PANCI cells were lysed and prepared for immunoblotting to detect protein expression of the YAP target, CTGF. Blots are representative of at least three independent experiments.

[0053] FIGS. 17A-17C. Disrupting FN-binding integrins prevents the ability of CAFs to enhance tumor initiation. FIG. 17A. CAFs enhance tumor initiation via FN and FN-binding integrins (subcutaneous xenograft model). PANCI human PDAC cells were injected subcutaneously (with or without CAF-1299) to the flank areas of nu / nu mice. Mice were monitored twice weekly to detect the earliest emergence of palpable tumors. Graph shows tumor take rate vs. time for 10-12 mice per group, using a volume of 100mm3(computed as length x width2) as the threshold for tumor take. At the endpoint of the experiment (day 56), tumors were harvested and prepared for histological analysis. FIG. 17B. Bispecific antibody treatment reduces tumor stroma and increases necrosis. Tumors sections were stained using H&E. Areas of stroma (S) and necrosis (N) are noted. FIG. 17C. Bispecific antibody treatment reduces tumor stiffness. Cryosections of tumors were analyzed using atomic force microscopy to evaluate tissue stiffness. Dots depict the mean value for each 20 pm x 20 pm region of interest (ROI). 15 ROIs were evaluated per tumor, for 2 tumors per group.

[0054] FIGS. 18A-18D. ABT601 treatment reduces fibrotic effect of human CAFs coinjected with tumor cells. FIGS. 18A-18D. ABT601 -treated tumors contain less CAF -produced fibronectin, fibrosis, and angiogenesis. Serial FFPE sections of tumors were processed for immunohistochemical detection of human-specific FN (hFN) (FIG. 18A) or CD31 (FIG. 18D), indicated with arrow. Trichrome (FIG. 18B) and picrosirius red (FIG. 18C) histological stains were used to visualize collagen. Graphs depict the quantification of H4C staining using QuPath, with each dot representing the mean value for each tumor slice examined. P-values were computed using Student’ s t-test.

[0055] FIG. 19. CAF-produced fibronectin boosts tumor initiation (summary schematic). Cancer-associated fibroblasts produce a dense and reactive stroma that supports tumor initiation and progression. The results herein establish the link between cell surface integrins and FN as a lynchpin for the construction of pro-tumor ECM that can account for the ability of CAFs to support tumor initiation for multiple in vitro and in vivo models. Knockdown or antibody blockade of integrins avP3 and / or a5pi can disrupt FN fiber assembly. FN fibers are known to function as a scaffold that mediates the assembly of other ECM proteins and incorporation of pro-tumor secreted factors. Knockdown of avP3 / a5pi or their dual blockade using a novelbispecific antibody (ABT601) can disrupt this cascade by preventing the initial assembly of FN fibers. This approach has broad use as a novel strategy to target the aberrant fibrosis that exacerbates the progression of cancer and fibrotic disease.

[0056] FIGS. 20A-20D. ABT601 reduces fibrotic response in metabolic dysfunction- associated steatohepatitis (MASH) model. (FIG. 20A) Primary liver cells extracted from human donor were combine with ABT601 and allowed to form spheroids for 7 days before stimulated with a MASH cocktail. At day 14, spheroids and supernatant were harvested and assayed. (FIG. 20B) Bright field images were taken on day 14 and spheroid diameter measured and normalized using ImageJ software. (FIG. 20C) ELISA was performed on spheroid supernatant to measure protein concentration of Collagen I. (FIG. 20D) qPCR was performed on spheroid lysates for genes important in fibrotic response.DETAILED DESCRIPTION

[0057] The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0058] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0060] Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant and synthetic polypeptides, antibodies or antigenbinding fragments thereof, manipulation of nucleic acid sequences, and production of transformed cells. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Ed. (Cold Spring Harbor, NY); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).

[0061] All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entirety.Definitions

[0062] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0063] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0064] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0065] Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0066] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0067] As used herein, ranges can be expressed as from “about” one particular value, and / or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0068] The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0069] The term “consists essentially of’ (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and / or 3’ or N-terminal and / or C-terminal ends of the recited sequence or between the two ends (e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added together.

[0070] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0071] The term “chimeric” refers to a molecule having two or more portions that are not naturally found together in the same molecule.

[0072] A “nucleic acid” or “nucleotide sequence” is a sequence of nucleotide bases, and may be RNA, DNA, or DNA-RNA hybrid sequences (including both naturally occurring and non- naturally occurring nucleotide) but is preferably either single or double stranded DNA sequences.

[0073] As used herein, the term “isolated” means a molecule, e.g., a protein, polynucleotide, or cell, separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell structural components or other polypeptides or nucleic acids commonly found associated with the molecule. The term also encompasses molecules that have been prepared synthetically.

[0074] By the terms “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms) it is meant that the severity of the subject's condition is reduced or at least partially improved or ameliorated and / or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and / or there is a delay in the progression of the condition.

[0075] As used herein, the terms “prevent,” “prevents,” or “prevention” and “inhibit,” “inhibits,” or “inhibition” (and grammatical equivalents thereof) are not meant to imply complete abolition of disease and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset of the condition, and / or reduces the symptomsassociated with the condition after onset. Inhibition can comprise a reduction of the incidence of a 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 of the onset of a condition can comprise a delay 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.

[0076] An “effective,” “ prophy lactically effective,” or “therapeutically effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, an “effective,” “prophylactically effective,” or “therapeutically effective” amount is an amount that will provide some delay, alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the effects need not be complete or curative, as long as some benefit is provided to the subject.

[0077] As used herein, a composition “targeting fibronectin” refers to a composition capable of inhibiting binding of fibronectin to one or more cognate receptors on the surface of a cell. A composition targeting fibronectin may directly block or interfere with the binding interaction between fibronectin and one or more of its cognate receptors or inhibit expression of fibronectin or one or more of its cognate receptors. In some embodiments, the one or more cognate receptors of fibronectin are selected from the group of avP3, avP6, and a5pi.

[0078] As used herein, the term “bind specifically” or “specifically binds” in reference to a bispecific antibody, or antigen-binding fragment thereof, of the invention means that the agent will bind with an epitope (including one or more epitopes) of a target but does not substantially bind to other unrelated epitopes or molecules. In certain embodiments, the term refers to an agent that exhibits at least about 60% binding, e.g., at least about 70%, 80%, 90%, or 95% binding, to the target epitope (e.g., avP3, avP6, a5pi) relative to binding to other unrelated epitopes or molecules. The term “specifically binds” does not necessarily require that an agent binds exclusively to its intended target. Rather, an agent specifically binds if its affinity for its intended target is about 2-fold greater when compared to its affinity for an unrelated epitope or molecule. Preferably the affinity of an agent will be at least about five-fold, e.g., at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In some embodiments, specific binding between a bispecific antibody, or antigen-binding fragment thereof, and an antigen means a binding affinity of at least about 106M e.g., at least about 107M or at least about 108M1to about 109M1or 1010M"1.

[0079] As used herein, the term “gene interference” or “gene expression interference” means the delivery of nucleic acid to the cell for sole purposes of inhibition of gene expression through mRNA interaction.

[0080] As used herein, the term “isolation stress” means the microenvironmental stresses encountered by solitary tumor cells that are not surrounded by other tumor cells, but rather their immediate environment contains normal cells and extracellular matrix as opposed to the highly reactive tumor stroma or areas with immune cell / vascular infiltrates typically seen in an established tumor. Examples of tumor cells experiencing isolation stresses include tumorinitiating cells at the primary and metastatic sites, dormant tumor cells in normal tissues, circulating tumor cells in the blood / lymphatics, and single tumor cells undergoing migration / invasion. These microenvironmental stresses include, but are not limited to, the loss of cell-cell contact, detachment from the appropriate matrix, hypoxia, nutrient deprivation, and other related ones.Bispecific antibody, or antigen-binding fragment thereof

[0081] A first aspect of the invention relates to a bispecific antibody, or antigen-binding fragment thereof, comprising two different variable domains that bind two different integrins selected from a5P 1, av33, and avP6. In some embodiments, the present invention relates to a bispecific antibody, or antigen-binding fragment thereof, comprising a first variable domain that specifically binds to integrin avP3 or integrin avP6 and a second variable domain that specifically binds to integrin a5pi or integrin avP6, wherein the first variable domain and the second variable domain are different, z.e., bind two different integrins. In some embodiments, a bispecific antibody, or antigen-binding fragment thereof, comprises a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin a5pi. In some embodiments, a bispecific antibody, or antigen-binding fragment thereof, comprises a first variable domain that specifically binds to integrin avP6 and a second variable domain that specifically binds to integrin a5pi. In some embodiments, a bispecific antibody, or antigen-binding fragment thereof, comprises a first variable domain that specifically binds to integrin avP3 and a second variable domain that specifically binds to integrin avP6.

[0082] The bispecific antibody, or antigen-binding fragment thereof, may be any structure that is capable of binding to two antigens on the same cell or different cells. In some embodiments, the bispecific antibody, or antigen-binding fragment thereof, exhibits improved binding to cells comprising the two antigens compared to binding of the constituent antibodiesused in the preparation of the bispecific antibody, or antigen-binding fragment thereof. In some embodiments, the variable domains of the bispecific antibody, or antigen-binding fragment thereof, synergistically bind to cells comprising the two antigens. As used herein, “synergistically,” “synergy,” or grammatical variations thereof, refers to binding and / or biological effect of a bispecific antibody, or antigen-binding fragment thereof, that is more than the additive sum of the binding and / or biological effect of the antibodies from which the variable domains of the bispecific antibody, or antigen-binding fragment thereof, were derived. In some embodiments, one or more portions of the bispecific antibody, or antigen-binding fragment thereof, are composed of antibody fragments.

[0083] In certain embodiments, the first domain of the bispecific antibody, or antigen-binding fragment thereof, is a first variable antibody domain. In certain embodiments, the second domain of the bispecific antibody, or antigen-binding fragment thereof, is a second variable antibody domain. In some embodiments, both domains are antibody domains. In some embodiments, the first domain is a humanized or human antibody domain. In some embodiments, the second domain is a humanized or human antibody domain. In some embodiments, the first domain and the second domain are humanized or human antibody domains. Example methods of making bispecific antibodies are described, for example, at scientistlive.com / content / bispecific-antibodies. In an embodiment, the CrossMab approach can be used to generate the bispecific antibody, which can comprise the crossover of antibody domains within one arm of a bispecific IgG antibody, for example, the constant CH I -CL domains or the variable heavy (VH) - variable light (VI.) domains within the Fab domain on one arm of a bi specific IgG antibody. The bispecific antibody can comprise further engineering strategies, including Knob in Hole (KiH) engineering of the Fc domain as described herein.

[0084] In some embodiments, the bispecific antibody or antigen-binding fragment thereof, further comprises an Fc domain of an IgG antibody. In an embodiment, the Fc domain is an Fc domain of an IgGl or IgG4 antibody. In an embodiment, the Fc domain is engineered to substitute a large amino acid for a smaller amino acid in the CH3 domain of one chain, and a small amino acid is substituted for a large amino acid in the opposite chain to engineer a KiH Fc domain. In some embodiments, the amino acid sequence of the bispecific antibody, or antigen-binding fragment thereof, comprises one or more of T363S, V366A, and / or Y404V (Eu numbering system) mutations. In some embodiments, the amino acid sequence of the bispecific antibody, or antigen-binding fragment thereof, comprises a T366W (Eu numbering system) mutation in the Fc domain of the second heavy chain of the bispecific antibody.

[0085] In some embodiments, the first domain and the second domain specifically bind integrins avP3, avP6 and / or a5pi on the surface of the cell, e.g., a cancer cell, e.g., an epithelial cancer cell. In some embodiments, the first domain and the second domain specifically bind integrins avP3 and a5pi on the surface of the cell, e.g, a cancer cell, e.g, an epithelial cancer cell. In some embodiments, the first domain and the second domain specifically bind integrins avP6 and a5pi on the surface of the cell, e.g., a cancer cell, e.g., an epithelial cancer cell. In some embodiments, the first domain and the second domain specifically bind integrins avP3 and avP6 on the surface of the 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 mesenchymal-like tumor cells. In some embodiments, the first variable domain specifically binds to integrin avP3. In some embodiments, the second variable domain specifically binds to integrin a5pi .

[0086] In some embodiments, the first domain and / or the second domain binds to an antigen that is not present, present at low levels, or present at lower levels on the surface of normal epithelial cells relative to epithelial cancer cells. In some embodiments, the antigen may be one that is not present, present at low levels, or present in reduced levels on the surface of epithelial cancer cells relative to an epithelial cancer cell beginning to transition to a mesenchymal cell. In some embodiments, the antigen may be one that is present or present at increased levels only after the epithelial cancer cell begins to transition to a mesenchymal cell. In some embodiments, the antigen is a mesenchymal cell antigen that is not present or only present at low levels on the epithelial cancer cell until it begins to transition to a mesenchymal cell.

[0087] In certain embodiments, the first domain comprises, consists essentially of, or consists of a Fab domain of an antibody. The Fab domain may be from any antibody isotype. In some embodiments, the first domain comprises a Fab domain of an IgG antibody, e.g., an IgGl 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 the CL of the light chain, and a heavy chain comprising the VL of the light chain and the CHI of the heavy chain of etaracizumab. In some embodiments, the first domain comprises a heavy chain VH-(AS)-hk.LC.h4.HC.S228P.T366W comprising the amino acid sequence of SEQ ID NO:1 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value therebetween), and a light chain VL-(SS)-hlv2.CHl comprising the amino acid sequence of SEQ ID NO:2 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value therebetween).

[0088] In certain embodiments, the second domain comprises, consists essentially of, or consists of a Fab domain of an antibody. The Fab domain may be from any antibody isotype. In some embodiments, the second domain comprises a Fab domain of an IgG antibody, e.g., an IgGl 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 the CHI of the light chain, and a heavy chain comprising the VL of the light chain and the CL of the light chain of volociximab and may optionally comprise one or more mutations in the Fc domain of the heavy chain. In some embodiments, the second domain comprises a heavy chain VH- h4.HC.S228P.T363S.V366A.Y404V comprising the amino acid sequence of SEQ ID NO:3 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value therebetween), and a light chain VL-hk.LC comprising the amino acid sequence of SEQ ID NO:4 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value therebetween).

[0089] In certain embodiments, the antibody comprises is a chimeric monoclonal antibody that specifically binds to and inhibits the functional activity of a5pi integrin. In some embodiments, the antibody comprises a heavy chain VH-h4.HC.S228P comprising the amino acid sequence of SEQ ID NO:5 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value therebetween), and a light chain VL-hk.LC comprising the amino acid sequence of SEQ ID NO:6 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto (or any range or value therebetween).

[0090] The bispecific antibody, or antigen-binding fragment thereof, may include sequence modifications that are known to enhance the characteristics of an antibody, e.g., stability, or alter the binding of the antibody to Fc-gamma receptors. In some embodiments, the sequence modifications provide selective cell engagement, for example, NK cell engagement, macrophage cell engagement, or no effector cell engagement. Thus, the properties of therapeutic antibodies can be modified to either enhance or suppress engagement with certain types of immune effector cells using a growing arsenal of glycoengineering and Fc engineering approaches (Saxena & Wu (2016) Front. Immunol. 7:580). In an embodiment, the sequence modifications provide variants that promote binding of the Fc portion of a therapeutic antibody to FcyRIIIA (CD16A), the only Fc receptor expressed on NK cells (Lazar et al. (2006) AppL Biol. Sci. 103(11):4005-4010). In some embodiments, the sequence modifications compriseenhanced binding to macrophages, including a G236A Fc mutant that promotes binding to FcyRIIA (CD32A) (Richards et al. (2008) Mol. Cancer Ther. 7(8):2517-27) or a bispecific antibody that recruits macrophages via FcaRI (CD89) (Li et al. (2018) OncoImmunoL 7(l):el380142).

[0091] In some embodiments, the amino acid sequence of the bispecific antibody, or antigenbinding fragment thereof, comprises a S228P (Eu numbering system) mutation in the hinge region. In some embodiments, the amino acid sequence comprises a S228P mutation in the Fc domain of an IgG e.g., IgGl, IgG4) antibody and allows for selective macrophage engagement. In some embodiments, the amino acid sequence comprises L234A, L235A, P329G (LALA-PG) mutation in the Fc domain of an IgG, (e.g., IgGl, IgG4) antibody and provides no effector cell engagement. In some embodiments, the amino acid sequence comprises a mutation selected from: a) S239D / A330L / I332E; b) I332E; c) G236A / S239D / I332E; d) G236A; e) N297A / E382V / M428I; f) M252Y / S254T / T256E; g) Q295R / L328 W / A330 V / P331 A / I332 Y / E382 V / M428I; h) L234A / L235A / P329G; i) M428L / N434S; j) L234A / L235A / P331S; k) L234A / L235A / P329G / M252Y / S254T / T256E; l) S298A / E333A / K334 / A; m) S239D / I332E; n) G236A / S239D / A330L / I332E; o) S239D / I332E / G236A; p) L234Y / G236W / S298A; q) F243L / R292P / Y300L / V305I / P396L; r) K326W / E333S; s) K326A / E333A; t) K326M / E333S; u) C221D / D222C; v) S267E / H268F / S324W;w) H268F / S324W; x) E345R y) R435H; z) N434A; aa) M252Y / S254T / T256E; ab) M428L / N434S; ac) T252L / T / 253S / T254F; ad) E294delta / T307P / N434Y; ae) T256N / A378V / S383N / N434Y; af) E294delta ag) L235E; ah) L234A / L235A; ai) S228P / L235E; aj) P331S / L234E / L225F; ak) D265A; al) G237A; am) E318 A; an) E233P; ao) G236R / L328R; ap) H268Q / V309L / A330S / P331S; aq) L234 A / L235 A / G237 A / P238 S / H268 A / A330 S / P331 S ; ar) A330L; as) D270A; at) K322A; au) P329A; av) P331A; aw V264A; ax) F241A; ay) N297A or G or N az) S228P / F234A / L235A; or ba) any combination of a) to az);(Eu numbering system) with or without the S228P mutation.

[0092] The following discussion is presented as a general overview of the techniques available for the production of antibodies; however, one of skill in the art will recognize that many variations upon the following methods are known.

[0093] The term “antibody” or “antibodies” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibody can be monoclonal, oligoclonal, or polyclonal and can be of any species of origin, including (for example) mouse, rat, hamster, rabbit, horse, cow, goat, sheep, pig, camel, monkey, or human, or can be a chimeric or humanized antibody. See, e.g., Walker et al. (1989) Mol. Immunol. 26:403. The antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also be chemically constructed according to the method disclosed in U.S. Pat. No. 4,676,980.

[0094] 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. Such fragments can be produced by known techniques. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 254: 1275). In some embodiments, the term “antibody fragment” as used herein may also include any protein construct that is capable of binding a target antigen.

[0095] Antibodies of the invention may be altered or mutated for compatibility with species other than the species in which the antibody was produced. For example, antibodies may be humanized or camelized. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions (z.e., the sequences between the CDR regions) are those of a human immunoglobulin consensus sequence. The humanized antibody can be a superhumanized antibody where only two CDRs are non-human (U.S. Pat. No. 7,087,409). The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. (1986) Nature 321 :522; Riechmann et al. (1988) Nature 332:323; Presta (1992) Curr. Op. Struct. Biol. 2:593).

[0096] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can essentially be performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321 :522; Riechmann et al. (1988) Nature 332:323; Verhoeyen et al. (1988) Science 239: 1534), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues (e.g., all of the CDRs or a portion thereof) and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.

[0097] Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom & Winter (1991) J. Mol. Biol. 227:381; Marks et al. (1991) J. Mol. Biol. 222:581). The techniques of Cole et al. and Boerner et al. are also available for the preparation of 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). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: 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.

[0098] Immunogens (antigens) are used to produce antibodies specifically reactive with target polypeptides. Recombinant or synthetic polypeptides and peptides, e.g., of at least 5 (e.g., at least 7 or 10) amino acids in length, or greater, are the preferred immunogens for the production of monoclonal or polyclonal antibodies. In one embodiment, an immunogenic polypeptide conjugate is also included as an immunogen. The peptides are used either in pure, partially pure or impure form. Suitable polypeptides and epitopes for target pathogens and sperm are well known in the art. Polynucleotide and polypeptide sequences are available in public sequence databases such as GENBANK® / GENPEPT®. Large numbers of antibodies that specifically bind to target cancer cell antigens have been described in the art and can be used as starting material to prepare the antibodies of the present invention. Alternatively, new antibodies can be raised against target antigens using the techniques described herein and well known in the art.

[0099] Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells and purified using standard techniques. The polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide.

[0100] Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, e.g., a purified or synthetic peptide, a peptide coupled to an appropriate carrier (e.g., glutathione-S-transferase, keyhole limpet hemocyanin, etc.), or a peptide incorporated into an immunization vector such as a recombinant vaccinia virus is optionally mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the peptide of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the peptide is performed where desired. Antibodies, including binding fragments and single chain recombinant versions thereof, against the polypeptides are raised by immunizing animals, e.g., using immunogenic conjugates comprising a polypeptide covalently attached (conjugated) to a carrier protein as described above. Typically, the immunogen of interest is a polypeptide of at least about 10 amino acids, in another embodiment the polypeptide is at least about 20 amino acids in length, and in another embodiment, the fragment is at least about 30 amino acids in length. Theimmunogenic conjugates are typically prepared by coupling the polypeptide to a carrier protein (e.g., as a fusion protein) or, alternatively, they are recombinantly expressed in an immunization vector.

[0101] Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified peptides or screened for agonistic or antagonistic activity. Specific monoclonal and polyclonal antibodies will usually bind with a KD of at least about 50 mM, e.g., at least about 1 mM, e.g., at least about 0.1 mM or better. In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as rodents, lagomorphs, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in Kohler & Milstein ((1975) Nature 256:495- 497). Summarized briefly, this method proceeds by injecting an animal with an immunogen, e.g., an immunogenic peptide either alone or optionally linked to a carrier protein. The animal is then sacrificed, and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or “hybridoma” that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.

[0102] Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host. The polypeptides and antibodies of the present invention are used with or without modification and include chimeric antibodies such as humanized murine antibodies. Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246: 1275-1281; Ward et al. (1989) Nature 341 :544-546.

[0103] Antibodies specific to the target polypeptide can also be obtained by phage display techniques known in the art.

[0104] The present invention additionally provides polynucleotides encoding the bispecific antibody, or antigen-binding fragment thereof, of this invention.

[0105] Further provided herein is a vector comprising the polynucleotide of the invention. Vectors include, but are not limited to, plasmid vectors, phage vectors, virus vectors, or cosmid vectors.

[0106] In some embodiments, the present invention provides a host cell comprising the polynucleotide and / or vector of this invention. The host cell can be a eukaryotic or prokaryotic cell and may be used for expressing the bispecific antibody, or antigen-binding fragment thereof, or other purposes.

[0107] A further aspect of the invention relates to a composition comprising the bispecific antibody, or antigen-binding fragment thereof, of the invention and a carrier. In some embodiments, the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

[0108] In some embodiments, the pharmaceutical composition may further comprise an additional therapeutic agent, e.g., a chemotherapeutic agent. Agents useful for treating cancer include, without limitation: 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 agents of the invention are administered in conjunction with anti-angiogenesis agents, such as antibodies to VEGF (e.g., bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®)) and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1), antibodies to 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 (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, and celecoxib, or any combination thereof. In other embodiments, the agents of the invention are administered in conjunction with one or more therapeutic antibodies, e.g., anti-cancer antibodies or antibodies to immune checkpoints. In other embodiments, the agents of the invention are administered in conjunction 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, 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 the immune checkpoint protein. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1, e.g., an antibody that specifically binds 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 antigen-binding fragment thereof, may be directly or indirectly linked with an additional therapeutic agent to form an antibody drug conjugate.

[0109] An additional aspect of the invention relates to a kit comprising the bispecific antibody, or antigen-binding fragment thereof, of the invention or cells for producing the bispecific antibody, or antigen-binding fragment thereof, of the invention. In some embodiments, the kit can include multiple bispecific antibodies, or antigen-binding fragments thereof and / or compositions containing such antibodies or antigen-binding fragments thereof. In some embodiments, the kit can further include an additional active agent, e.g., a chemotherapeutic agent as would be known to one of skill in the art. In some embodiments, the kit can further include additional reagents, buffers, containers, etc.Methods using an antibody that binds integrins or an antigen-binding fragment thereof

[0110] One aspect of the invention relates to a method of preventing the binding of a cell to fibronectin, comprising contacting the cell with an effective amount of an antibody or antigenbinding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6 and / or integrin a5pi. The method can comprise preventing the binding of the cell to fibrillar fibronectin, globular fibronectin, or both fibrillar fibronectin and globular fibronectin. Fibronectin structure and domains are described in Dalton & Lemmon ((2021) Cells 10(9): 2443; Figure 1, incorporated herein by reference in its entirety). Fibronectin has been related to chemotherapy resistance in cancer, including cisplatin resistance. See, e.g., Gao et al. (2016) Biochem. Biophys. Res. Commun. 476(1):35-41 ; Spada et al. (2021 ) . / . Exp. Clin.Cancer Res. 40: 102. Increased fibronectin is also implicated in fibrotic diseases. See, e.g., Van Vliet et al. (2001) J. Pathol. 193(2):256-262. Integrin subunit av is present on many cells, including fibroblasts, epithelial cells, and neural tissues. Integrin subunits P3, a5, and P6 are mesenchymal-like markers; however, cellular stress induces expression of integrin subunits P3, a5, and P6 on epithelial-like cancer cells. Enrichment of integrins such as a5pi, avP3, avP6, or a6p4 are observed on tumor-initiating, metastasizing, or stress-resistant cells. In some embodiments, the method comprises contacting the cell with an effective amount of the bispecific antibody, or antigen-binding fragment thereof, of the invention. In some embodiments, the method comprising contacting a cell with an effective amount of a bispecific antibody, or antigen-binding fragment thereof, of the present invention targeting more than one fibronectin-binding integrin prevents binding across a heterogeneous tumor population, for example, when antigens are recognized on different tumor cells; increases or enhances recognition of tumor cells, killing of tumor cells, and / or inhibiting of metastasis or growth of tumor cells when both antigens are recognized on a single cell; and / or homes to the tumor more effectively. In some embodiments, the tumor is a fibrotic tumor and comprises stromal cells. [OHl] In some embodiments, the methods and compositions disclosed herein prevent or substantially reduce binding of fibronectin to a cancer cell 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%) as compared to binding of fibronectin to the cancer cell at the primary tumor site in the absence of said composition. In some embodiments, the methods and compositions disclosed herein prevent or substantially reduce binding of fibronectin to a cell involved in the seeding, migration and / or invasion of a tissue by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) as compared to binding of fibronectin to the same cell in the absence of said composition. In some embodiments, the methods and compositions disclosed herein prevent or substantially reduce binding of fibronectin to a cell involved in fibrosis by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) as compared to binding of fibronectin to the same cell in the absence of said composition. In some embodiments, the methods and compositions disclosed herein prevent or substantially reduce fibronectin and collagen fibril formation by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) as compared to fibronectin and collagen fibril formation in the absence of said composition. In some embodiments, the composition of the invention is a bispecific antibody, or antigen-binding fragment thereof, as described herein, and the bispecific antibody, or antigen-binding fragment thereof, exhibits at least about a 10% (least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100%) improvement in blocking binding of fibronectin to a cancer cell at a primary tumor site, reducing binding of fibronectin to a cell involved in the seeding, migration and / or invasion of a tissue, reducing binding of fibronectin to a cell involved in fibrosis, and / or reducing fibronectin and collagen fibril formation, as compared to either antibody from which the bispecific antibody, or antigen-binding fragment thereof, was derived.

[0112] One aspect of the invention relates to a method of treating a cancer expressing integrin, e.g., avP3 and / or integrin a5pi, in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3 and / or integrin a5pi. In some embodiments, the method of treating a cancer expressing integrin avP3 and / or integrin a5pi in a subject in need thereof comprises comprising administering a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof, to the subject, thereby treating the cancer.

[0113] One aspect of the invention relates to a method of treating a cancer in a subject in need thereof, comprising the steps of: a) selecting a subject having cancer cells that are enriched for integrin avP3, integrin avP6, and / or integrin a5pi, and optionally immune effector cells (e.g., macrophages); and b) administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi. In some embodiments, the method comprises administering a therapeutically effective amount of a bispecific antibody or antigenbinding fragment thereof, or a pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof, to the subject, thereby treating the cancer. In some embodiments, step a) comprises obtaining a sample of the cancer from the subject and measuring the level of integrin, e.g., integrin avP3, integrin avP6, and / or integrin a5pi, and optionally immune effector cells in the sample.

[0114] A further aspect of the invention relates to a method of treating a cancer expressing an antigen recognized by an antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example integrin avP3, integrin avP6, and / or integrin a5pi, in a subject in need thereof, comprising administering a therapeutically effective amount of antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, or the pharmaceutical composition of the invention to the subject, thereby treating the cancer. In some embodiments, the method of treating a cancer expressing an antigen recognized by the bispecific antibody, or antigen-binding fragment thereof, of the invention e.g., integrin avP3, integrin avP6, and / or integrin a5pi) in a subject in need thereof, comprises administering a therapeutically effective amount of the bispecific antibody, or antigen-binding fragment thereof, or the pharmaceutical composition of the invention to the subject, thereby treating the cancer

[0115] An additional aspect of the invention relates to a method of treating an epithelial cell cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, or the pharmaceutical composition of the invention to the subject, thereby treating the epithelial cell cancer. In some embodiments, the method of treating an epithelial cell cancer in a subject in need thereof, comprises administering a therapeutically effective amount of the bispecific antibody, or antigen-binding fragment thereof, or the pharmaceutical composition of the invention to the subject, thereby treating the epithelial cell cancer.

[0116] Another aspect of the invention relates to a method of treating a cancer in a subject in need thereof, comprising the steps of: a) selecting a subject having cancer cells that are enriched for an antigen specifically bound by an antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi and enriched for myeloid-derived cells; and b) administering a therapeutically effective amount of the antibody or antigenbinding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, or the pharmaceutical composition of the invention to the subject, thereby treating the cancer.

[0117] In some embodiments, the method of treating a cancer in a subject in need thereof, comprises the steps of: a) selecting a subject having cancer cells that are enriched for an antigen specifically bound by bispecific antibody, or antigen-binding fragment thereof, of the invention (e.g., integrin avP3) and enriched for myeloid-derived cells; and b) administering a therapeutically effective amount of the bispecific antibody, or antigen-binding fragment thereof, or the pharmaceutical composition of the invention to the subject, thereby treating the cancer.

[0118] The epithelial cancer may be any known type of carcinoma, for example, a squamous cell carcinoma, adenocarcinoma, transitional cell carcinoma, or basal cell carcinoma. In some embodiments, the cancer that is linked to chronic inflammation-related fibrosis. See, e.g.,Piersma et al. (2020) Biochim. Biophys. Acta Rev. Cancer 1873(2): 188356. Examples of epithelial cancers include, without limitation, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, renal cancer, breast cancer, brain cancer (e.g., glioma, e.g., glioblastoma multiforme), skin cancer, stomach cancer, prostate cancer, ovarian cancer, esophageal cancer, or colorectal cancer. In some embodiments, the epithelial cancer cell is a late-stage epithelial cancer cell. In some embodiments, the epithelial cancer cell has at least partially transitioned to a mesenchymal cell, e.g., expresses one or more mesenchymal antigens. In certain embodiments, the cancer cell is chemotherapy resistant or refractory, which may be due to the epithelial-to-mesenchymal transition.

[0119] In some embodiments, the methods may further comprise the step of isolating myeloid-derived cells from the subject, contacting the myeloid-derived cells with the antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, optionally a bispecific antibody, or antigenbinding fragment thereof, or pharmaceutical composition, and administering the contacted myeloid-derived cells to the subject.

[0120] In some embodiments, the cancer is an 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 multiforme), skin cancer, stomach cancer, prostate cancer, esophageal cancer, or colorectal cancer. The epithelial cancer may be any known type of carcinoma. Examples of epithelial cancers include, without limitation, cancers of the gastrointestinal tract, breast, lungs (e.g., non-small cell lung cancer), colon, prostate, or bladder. In some embodiments, the epithelial cancer cell is a late-stage epithelial cancer cell. Late stage or advanced stage, as used herein, refers to stage III or stage IV cancers based on the TNM staging system. In some embodiments, the epithelial cancer cell has at least partially transitioned to a mesenchymal cell, e.g., expresses one or more mesenchymal antigens. Cancer-associated fibroblasts, macrophages, and other immune cells secrete a variety of cytokines and factors that engage tumor cells to activate the expression of transcription factors that induce EMT. Mesenchymal- like carcinoma cells also shift the immune component of the tumor toward an immunocompromised state that excludes anti-tumor immune cell types and recruits pro-tumor macrophages. In certain embodiments, the epithelial cancer cell is chemotherapy resistant or refractory, which may be due to the epithelial-to-mesenchymal transition. In some embodiments, the cancer comprises a KRAS mutation (see, e.g., Huang et al. (2021) Signal Transduct. Targeted Ther. 6:386) or an EGFR mutation (see, e.g., Sigismund et al. (2018) Mol. Oncol. 12(1): 3-20.

[0121] In some embodiments, more than one antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, e.g., a bispecific antibody, or antigen-binding fragment thereof, may be delivered to a subject. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, may engage multiple targetable antigens.

[0122] Another aspect of the invention relates to a method of treating a fibrotic disorder comprising cells expressing integrin, for example, integrin avP3, integrin avP6, and / or integrin a5pi, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, optionally a bispecific antibody or antigen-binding fragment thereof, of the invention or a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3 and / or integrin a5pi, of the invention, thereby treating the fibrotic disorder. In some embodiments, the fibrotic disorder is eye fibrosis, heart fibrosis, hepatic fibrosis, intestinal fibrosis, lung fibrosis, pancreas fibrosis, renal fibrosis, non-alcoholic steatohepatitis, systemic sclerosis, idiopathic pulmonary fibrosis, fibromyalgia, keloids, or skin fibrosis. In some embodiments, the cell is a mesenchymal cell, a fibroblast, or a macrophage.

[0123] Another aspect of the invention comprises methods of inhibiting collagen organization in a subject, comprising 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 the one or more fibronectin receptor. In some embodiments, inhibiting the expression and / or function of the one or more fibronectin receptor inhibits collagen organization but does not affect adhesion.

[0124] Another aspect of the invention relates to a method of reversing collagen organization in a subject, comprising 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 the one or more fibronectin receptor.

[0125] In some embodiments, the composition comprises an antisense oligonucleotide, an RNAi, or an siRNA. In some embodiments, the composition comprises one or more antisense oligonucleotide, RNAi, or siRNA. In some embodiments, the composition targets an integrin,for example 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 avP3 and / or integrin a5pi.

[0126] A RNAi therapeutic comprises a polynucleotide that is complementary to a portion of the target sequence mRNA, generally ranging in size from 15 to about 50 base pairs. In an example embodiment, the siRNA is a nucleic acid that can form a double stranded RNA with the ability to reduce or inhibit expression of a gene or target gene: each complementary sequence of the double stranded siRNA is about 15 to about 50 nucleotides in length, and the double stranded siRNA is about 15 to about 50 base pairs in length. A small hairpin RNA (shRNA) is also contemplated for use. The shRNA is an antisense strand of about 19 to about 25 nucleotides followed by a short nucleotide loop (approximately 5 nt to 9 nt) followed by the analogous sense strand. In an embodiment, an RNAi is a microRNA or miRNA, endogenous RNAs, some of which are known to regulate the expression of protein-coding genes at the posttranscriptional level. See, e.g., Lim et al. (2003) Science 299: 1540; Lee & Ambros (2001) Science 294:862; Lau et al. (2001) Science 294:858-861; Lagos-Quintana et al. (2003) RNA 9: 175-179.

[0127] Different criteria are available for selecting the nucleic acid for use and may comprise scanning the mRNA sequence of the target, and may include empiric determination in accordance with, for example, Sui et al. ((2002) Proc. Natl. Acad. Sci. USA 99:5515-20), and may include confirmation the sequence lacks significant sequence homology with other genes as analyzed by BLAST search. Additional approaches may comprise any accessible site in endogenous mRNA can be targeted for degradation by synthetic oligodeoxyribonucleotide / RNase H method (see, e.g., Lee et al. (2002) Nature Biotechnol. 20:500-05). RNAi treatment may comprise miRNA or siRNA, or a pre-miRNA which is processed by Dicer to form a miRNA. The RNAi may also comprise a dsRNA or shRNA which is processed by Dicer to form a siRNA. The polynucleotides may comprise one or more modifications to suppress innate immune activation, enhance activity and specificity, and reduce off-target induced toxicity. Example teachings can be found, for example at Provost et al. (2002) EMBO J. 21(21):5864-5874; Tabara et al. (2002) Cell 109(7): 861 -71; Martinez et al. (2002) Cell 110(5):563; Hutvagner & Zamore (2002) Science 297:2056. In certain embodiments, a single-stranded RNAi agent disclosed herein can comprise substitutions, or modifications, including chemically modified nucleotides, and non-nucleotides which may include incorporation in the backbone, sugars, bases, or nucleosides. The use of substituted or modified single-stranded RNAi agents can be designed to have an increased half-life in asubject. Furthermore, certain substitutions or modifications can be used to improve the bioavailability of single-stranded RNAi agents by targeting particular cells or tissues or improving cellular uptake of the single-stranded RNAi agents. Exemplary modifications and locations within a RNAi polynucleotide are described in Hu et al. ((2020) Signal Transduct. Targeted Ther. 5: 100, incorporated herein by reference, see, e.g., Figures 2 and 3, specifically for its teachings of modifications).

[0128] In some embodiments, the composition comprises an antibody or antigen-binding fragment thereof that specifically binds to fibronectin. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to one or more integrin subunit, e.g., comprising an alpha and / or beta subunit. In some embodiments, the antibody or antigenbinding fragment thereof is monospecific, or the antibody or fragment thereof is the bispecific antibody, or antigen-binding fragment thereof as detailed elsewhere herein, for example, binding an integrin selected from integrin avP3, integrin avP6, and integrin a5pi.

[0129] Another aspect of the invention comprises methods of inhibiting expression of Yes- associated protein 1 (YAP) or YAP target genes in a subject, comprising administering antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, optionally a bispecific antibody, or antigen-binding fragment thereof, of the present invention, a polynucleotide encoding the antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, optionally a bispecific antibody, or antigen-binding fragment thereof, or a vector comprising the polynucleotide to the subject. In some embodiments, YAP expression or YAP target gene expression is inhibited by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) as compared to YAP expression or YAP target gene expression in the absence of the antibody, or antigen-binding fragment thereof. In an embodiment, the YAP target gene is CTGF or CYR61. See, e.g., Pobbati et al. (2020) Theranostics 10(8):3622-3635. In some embodiments, inhibiting expression of YAP protein and / or YAP target genes reduces growth of solid tumors, inhibits cancer, reduces oncogenic processes, such as sternness, drug resistance, immune suppression by at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) as compared to the growth of solid tumors, growth of cancer, or oncogenic processes in the absence of the antibody, or antigen-binding fragment thereof.

[0130] The methods of the invention may further comprise administering to the subject an additional therapeutic agent or treatment (e.g., surgery, radiation). Fibrotic therapeutics include anti-fibrotic agents, for example nintedanib, pirfenidone, saracatinib, FG-3019, bardoxolonemethyl, apabetalone, angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs). Cancer therapeutic agents include, without limitation, 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). Other cancer therapeutic agents include, without limitation, anti-angiogenesis agents, such as antibodies to VEGF (e.g., bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®)) and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1), 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 (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, and methotrexate.

[0131] In some embodiments, the methods further comprise administering to the subject a CD47 blocking agent to enhance phagocytosis of the cancer cells. Such agents include CD47- blocking monoclonal antibodies (Hu5F9-G4, CC-90002, Ti-061, or SRF231) or SIRPa-Fc fusion proteins (TTI-621, TTI-622, ALX148). However, one of the advantages of the present invention is that the method is effective against cancers whether or not the cancer cells express CD47. Thus, in some embodiments, the methods of the invention are used to treat cancers that express CD47. In some embodiments, the methods of the invention are used to treat cancers that do not express CD47. In some embodiments, the methods of the invention do not comprise administering to the subject a CD47 blocking agent.

[0132] In some embodiments, the methods further comprise administering to the subject an immune checkpoint inhibitor. 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 the immune checkpoint protein. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4 that are enriched in mesenchymal tumors, e.g., an antibody that specifically binds PD-1, PD-L1, or CTLA-4. In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, ipilimumab, durvalumab, or atezolizumab.

[0133] In some embodiments, the methods further comprise administering to the subject an EGFR inhibitor. 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 methods further comprise administering to the subject a KRAS inhibitor, in particular, for patients with KRAS G12C mutation, which may be sotorasib, MRTX1133, or ARS1620.

[0134] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, optionally a bispecific antibody, or antigen-binding fragment thereof, used in the methods of the present invention is administered directly to a subject. In some embodiments, antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, will be suspended in a pharmaceutically acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or administered subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. In another embodiment, the intratracheal or intrapulmonary delivery can be accomplished using a standard nebulizer, jet nebulizer, wire mesh nebulizer, dry powder inhaler, or metered dose inhaler. The agents can be delivered directly to the site of the disease or disorder, such as lungs, kidney, or intestines, e.g., injected in situ into or near a tumor. The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient’s illness; the subject’s size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages for each agent are in the range of 0.01-100 pg / kg. Wide variations in the needed dosage are to be expected in view of the variety of agents available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can beadjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-, or more fold). Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or nanoparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.

[0135] By “pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, ie., the material can be administered to a subject without causing any undesirable biological effects such as toxicity.

[0136] The formulations of the invention can optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.

[0137] The antibody or antigen-binding fragment thereof that specifically binds one or more integrins, for example, integrin avP3, integrin avP6, and / or integrin a5pi, optionally a bispecific antibody, or antigen-binding fragment thereof, of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA). In the manufacture of a pharmaceutical formulation according to the invention, the agent is typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and may be formulated with the agent as a unit-dose formulation, for example, a capsule or vial, which can contain from 0.01 or 0.5% to 95% or 99% by weight of the agent. One or more agents can be incorporated in the formulations of the invention, which can be prepared by any of the well- known techniques of pharmacy.

[0138] The formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intrathecal, and inhalation administration, administration to the liver by intraportal delivery, as well as direct organ injection (e.g., into the liver, into the brain for delivery to the central nervous system, or into the pancreas) or injection into a body cavity. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular agent which is being used.

[0139] For injection, the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL® (BASF, Parsippany, NJ). For other methods of administration, the carrier can be either solid or liquid.

[0140] For oral administration, the agent can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Agents can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric- coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0141] Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the agent in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the agent in an inert base such as gelatin and glycerin or sucrose and acacia.

[0142] Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the agent, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations can be presented in unit / dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.

[0143] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising an agent of the invention, in a unit dosage form in a sealed container. The agent is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 1 mg to about 10 grams of the agent. When the agent is substantially water-insoluble, a sufficient amount of emulsifying agent which ispharmaceutically acceptable can be employed in sufficient quantity to emulsify the agent in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0144] Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by admixing the agent with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

[0145] Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0146] Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Tyle (19886) Pharm. Res. 3:318) and typically take the form of an optionally buffered aqueous solution of the compounds. Suitable formulations comprise citrate or bis / tris buffer (pH 6) or ethanol / water and contain from 0.1 to 0.2M of the compound.

[0147] The agent can alternatively be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the agent, which the subject inhales. The respirable particles can be liquid or solid. The term “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See 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. Aerosols of liquid particles comprising the agent can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising the agent can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.

[0148] Alternatively, one can administer the compound in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0149] Further, the present invention provides liposomal formulations of the agents disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles. In such an instance, due to the water solubility of the agent, the agent will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free. When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt can be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.

[0150] The liposomal formulations containing the agent can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.

[0151] In the case of water-insoluble agents, a pharmaceutical composition can be prepared containing the water-insoluble agent, such as for example, in an aqueous base emulsion. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the agent. Particularly useful emulsifying agents include phosphatidylcholines and lecithin.

[0152] In particular embodiments, the compound is administered to the subject in a therapeutically effective amount, as that term is defined above. Dosages of pharmaceutically active agents can be determined by methods known in the art, see, e.g., Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA). The therapeutically effective dosage of any specific agent will vary somewhat from agent to agent, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg / kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the agent. Toxicity concerns at the higher level can restrict intravenous dosages to a lower level such as up to about 10 mg / kg, with all weights being calculated based upon the weight of the agent. A dosage from about 10 mg / kg to about 50 mg / kg can be employed for oral administration. Typically, a dosage from about 0.5 mg / kg to 5 mg / kg can be employed for intramuscular injection. Particular dosages are about 1pmol / kg to 50 pmol / kg, and more particularly to about 22 pmol / kg and to 33 pmol / kg of the agent for intravenous or oral administration, respectively.

[0153] In particular embodiments of the invention, more than one administration (e.g, two, three, four, or more administrations) can be employed over a variety of time intervals (e.g, hourly, daily, weekly, monthly, etc.) to achieve therapeutic effects.

[0154] The present invention finds use in veterinary and medical applications. Suitable subjects include both avians and mammals, with mammals being preferred. The term “mammal” as used herein includes, but is not limited to, humans, primates, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, and adults. The subject may be one in need of the methods of the invention, e.g., a subject that has or is suspected of having cancer. The subject may be a laboratory animal, e.g., an animal model of a disease.

[0155] The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.EXAMPLESEXAMPLE 1: Materials and Methods

[0156] Reagents, Chemicals, and Commercial Antibodies. Primary antibodies used in this study include FN (E5H6X) (Cell signaling #26836, RRID: AB_2924220, 1 : 1000 for western blots, 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 blots; 1 :200 for immunofluorescence staining), CTGF (D8Z8U) (Cell Signaling #86641, RRID: AB_2800085, 1 : 1000 for western blots), vinculin (Boster #MA1103, RRID: AB 3082541, 1 : 15,000 for western blots), GAPDH (D16H11) (Cell Signaling #5174, RRID: AB_10622025, 1 :3000 for western blots), anti-a5 integrin antibody (P1D6) (EMB Millipore MAB1956Z, RRID: AB 94455, 10 pg / ml for flow cytometry), and CD31 (R&D Systems #AF3628, RRID:AB_2161028, 1 :40 for immunohistochemistry). Anti-avP3 integrin antibody (LM609) (10 pg / ml for flow cytometry) was purchased from Millipore (MAB1976, RRID: AB 2296419). Predesigned siRNAs used in this study were purchased from Millipore Sigma. Each siRNA combo is mixed with two distinct siRNAs (siRNAl and siRNA2, 1 : 1 mixture) targeting different gene regions of the gene of interest. The siRNA IDs are provided in Table 1.Table 1. siRNA IDs

[0157] Novel Antibodies. ABT101 (heavy chain ETAR.VH-h4.HC.S228P, SEQ ID NO:7; and light chain ETAR.VH-hk.LC, SEQ ID NO:8) and ABT701 (heavy chain VH- h4.HC.S228P, SEQ ID NO:5; and light chain VL-hk.LC, SEQ ID NO:6) are h!gG4-S228P monoclonal antibodies that recognize human integrins avP3 and a5pi, respectively (Table 2). ABT601 is a bispecific antibody designed for dual recognition of FN-binding integrins avP3 and a5pi. ABT601 is comprised of the antigen-recognizing Fab domains of ABT101 and ABT701 with the same h!gG4-S228P Fc domain.Table 2. Antibodies

[0158] Cells. PANCI human pancreatic cancer cells were obtained from the American Type Culture Collection (ATCC CRL-1469, RRID: CVCL 0480) and cultured using DMEM. Cell line authentication by short tandem repeat (STR) analysis was performed for the PANCI cells in April 2015 and November 2024. KP4 human pancreatic cancer cells were obtained, along with STR cell line authentication in January 2023, from the Riken BioResource Research Center (BRC) Cell Bank (RRID: CVCL 1338) and cultured using RPMI. Dr. Andrew Lowy(UC San Diego) provided low- passage stock vials of immortalized CAF cell lines hPCF1299 (CAF-1299) and hPCF1424 (CAF- 1424) in June 2022 that were previously derived (Hurtado de Mendoza et al. (2021) Nat. Commun. 12(1): 1541) from fresh surgical specimens of human PDAC tissue. All cells were expanded upon receipt, tested for mycoplasma using PCRto detect the 16S rRNA gene from the M. mycoides cluster (Forward primer: 5'-CGA AAG CGG CTT ACT GGC TTG TT-3' (SEQ ID NO:9); Reverse primer: 5'-TTG AGA TTA GCT CCC CTTCAC AG-3' (SEQ ID NO: 10)), and cryopreserved as low-passage stocks (Bascunana et al. (1994) J. Bacterial. 176(9):2577-86). For each experiment, stock vials were thawed, cultured for no more than 30 passages, and tested biweekly for mycoplasma.

[0159] Cell-Free ECM Generation and Cell-Based Assays. Cells were seeded in eight-well chamber slides, glass coverslips, or tissue-treated cell culture plates for up to 7 days to allow cells to produce and deposit sufficient ECM. Decellularization was performed as previously reported (Harris et al. (2018) Methods Cell. Biol. 143:97-114). Samples were washed twice with Hanks’ Balanced Salt Solution (HBSS), incubated for 15-20 minutes at 37°C in lysis buffer (8 mM Na2HPO4, 1% NP-40, pH 9.6), rinsed three times with wash buffer (10 mM Na2HPO4, 300 mM KC1, pH 7.5) and three times with sterile deionized water, then stored in HBSS. Cell-free ECM was then used for immunofluorescence staining or other cell-based assays. For cell-based assays, pancreatic cancer cells were seeded atop the cell- free ECM in the presence of 10% or 2% FBS-containing media and grown for 24 hours before cell harvest for western blots analysis.

[0160] Quantitative RT-PCR RNA was isolated using the RNeasy RNA Purification Kit (Qiagen, 75144) following 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 CFX96 (Bio-Rad) with SYBR® Green dye (Bio-Rad, 1725272). The custom primer sequences are provided in Table 3.Table 3. Primers

[0161] Mouse Study Approval. All experiments involving mice were conducted under protocol S05018, approved by the UC San Diego Institutional Animal Care and Use Committee. All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All animals were housed under standard conditions, i.e. given unrestricted access to food and water, housed in standard cages, and rooms regulated to control temperature and light cycles. Cell lines used for in vivo experiments confirmed negative for a panel of human pathogens.

[0162] Subcutaneous xenograft model. The number of PANCI cells injected varies greatly among studies (Kuninty et al. (2019) Set. Adv. 5(9):eaax2770; Watabe et al. (2020) J. Nucl. Med. 61 (4): 563-569; Wang et al.A) Pancreas 43(2):291-7), and this is likely influenced by the use of various strains of immune-compromised mice. Since nu / nu mice have a relatively less compromised immune system than NOD-SCID or NSG strains, we selected an injection amount of 0.5 million cells for the experiment in FIG. 17A. 5xl05human PANCI cells were mixed with or without an equal number of human CAF-1299 cells transfected with different siRNAs for 72 hours. Knockdown was verified via western blot.

[0163] For the antibody treatment groups, PANCI cells or CAF-1299 were pre-mixed with 10 pg / mL antibody for 10 minutes before injection. Cells were suspended in a 1 : 1 mixture of HBSS and Phenol Red-free Basement membrane matrix (BD Biosciences) (total volume is 100 pL per injection) and injected subcutaneously in 6-to-8-week-old female immune- compromised 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 examined twice weekly for palpable tumors. A tumor larger than 100 mm3in volume is counted.

[0164] Orthotopic Pancreatic Cancer Model. To determine an injection number that would allow us to assess changes in tumor initiation at a relatively early timepoint, we performed a pilot study to compare orthotopic injections of 5, 1, or 0.2 million KP4-luc cells. While no tumors were detected by bioluminescence imaging after 5 weeks, imaging at 10 weeks revealed tumors in 3 / 3 mice that received 5 or 1 million KP4-Luc cells but only 1 / 3 mice for the 0.2 million cell group. We reasoned that orthotopic implantation of 1 million KP4-Luc cells should produce tumors in all mice that are detectable after 5-10 weeks, and furthermore that coinjection with CAFs should accelerate tumor initiation (z.e., detection at timepoints less than 5 weeks). For the experiment shown in FIG. 13A, IxlO6human KP4 cells stably transfected with luciferase lentivirus (KP4-Luc) were mixed with or without the equal number of human CAF- 1299 cells transfected with different siRNAs for 72 hours. Knockdown was verified via western blot. Cells were suspended in HBSS and injected into the pancreas of 6-to-8-week-old femaleimmune-compromised nu / nu mice (Charles River Labs #088, RRID: IMSR_CRL:088). The tumor growth was monitored twice a week by using non-invasive bioluminescence imaging using an IVIS Spectrum system (Perkin-Elmer). All mice were imaged 10 minutes after being injected with D-luciferin (L9504, Sigma- Aldrich). Immunoblotting to assess the duration of siRNA-mediated knockdown of FN1 for CAFs showed that FN knockdown remained effective after 6 days in culture, but significantly declined by day 14.

[0165] Immunofluorescence Staining and Confocal Microscopy . CAFs were seeded on an eight-well chamber slide (Nuc Lab-Tek chamber slide, Thermo Scientific) overnight. The next day, cells were treated with siRNA targeting the genes of interest or antibodies (10 pg / mL) for 72 hours and then fixed with 4% paraformaldehyde for 15 minutes at room temperature. To target pre-existing matrix, CAFs were seeded and allowed to deposit matrix for 72 hours. At 72 hours, antibody was added (10 pg / ml) and allowed to incubate for an additional 72 hours. The cells and matrix were then fixed with 4% paraformaldehyde for 15 minutes at room temperature. All instances were followed by 45 minutes of incubation with a blocking buffer containing HBSS or HBSS-Tween® 20 (0.2%) supplemented with 5% horse serum. Cells were then incubated with primary antibodies at a dilution of 1 :200 (anti-FN or COL1A1) overnight at 4°C, followed by secondary antibodies at a dilution of 1 : 1000 for 1 hour and DAPI staining for 5 minutes at room temperature. Images were acquired by Nikon Eclipse Ti C2 confocal microscope with multiple Z- stack images and analyzed with NIS-Elements Viewer 5.21. The fluorescent signal was quantified as % area fraction using Imaged.

[0166] Immunoblotting. Immunoblotting was performed as previously described (Efthymiou et al. (2020) Front. Oncol. 10:641). Briefly, cells were washed twice with HBSS before lysing with either IX RIP A buffer containing protease and phosphatase inhibitors, 2X sample buffer containing IX reducing agent, or for conditioned supernatant, 4X sample buffer containing IX reducing agent (BioRad #1610737 and #1610747). A BCA assay (Thermo, 23227) was performed, and the lysates were normalized. Sample buffer (NuPAGE™ LDS Sample Buffer 4X, Sigma #NP0007) and reducing agent (NuPAGE™ Sample Reducing Agent, Sigma #NP0009) were added to the cell lysates. All samples were heated at 95°C for 5 minutes. 10 pg of protein or 30 pl of each sample containing Laemmli buffer were loaded onto an SDS- PAGE gel. Blocking was performed in 5% BSA in TBS-T, and probing was performed in 5% BSA in TBS-T buffer.

[0167] Flow Cytometry. Cell pellets were washed with PBS, blocked with 1% BSA in PBS for 30 minutes at room temperature, and stained with or without indicated primary antibodies with fluorescently labeled secondary antibodies. Cells were incubated with live / dead fixableblue dead cell stain kit (Invitrogen, L23105). Flow cytometry was performed on a BD Fortessa X-20 (BD) analyzer, and the data were analyzed using FlowJo (Treestar) software.

[0168] Immunohistochemistry. Immunohistochemical staining was performed on formalin- fixed paraffin-embedded slides using an ImmPRESS® Excel Staining Kit (Vector, MP-7602) following the manufacturer’s instructions. For integrin human FN, low-pH antigen retrieval (Invitrogen, 00- 4955-58) was performed for 40 minutes at 95°C. For CD31, high-pH antigen retrieval (Invitrogen, 00-4956-58) was performed for 40 minutes at 95°C. Trichrome staining was performed using Abeam AB 150686. Picrosirius Red staining was performed using Abeam AB246832. The slides were imaged using an Olympus VS200 Slide Scanner (Olympus) at The UCSD School of Medicine Microscopy Facility. Scanned images were analyzed for protein expression as area fraction per area of tumor tissue calculated using QuPath Open Software for Bioimage Analysis (Spada et al. (2021) J. Exp. Clin. Cancer Res. 40(1): 102).

[0169] Atomic Force Microscopy (AFM). AFM measurements were performed using an MFP-3D atomic force microscope (Asylum Research). Silicon nitride cantilevers were used with a normal spring constant of 0.08 Nm-1 and a 200 pm length (Nano World, PNP-TR-50). Cantilevers were calibrated using the thermal fluctuation method and verified by probing glass of known elasticity. The specimens used were 20 pm-thick OCT-embedded frozen human PDAC tissue sections that were thawed and equilibrated to room temperature by immersion in HBSS for 5 minutes. Indentation tests for the specimens were carried out at 2 pm per second loading rate to generate 16 force curves across equally distributed regions of 20 pm x 20 pm. The Young’s Moduli of the samples were determined by fitting force curves with the Hertz model using a Poisson ratio of 0.5.EXAMPLE 2: Blocking Fibronectin-Binding Integrins on Cancer-Associated Fibroblasts

[0170] CAF1299 cancer-associated fibroblast cells were treated with siRNAs for 72 hours and immunostained for fibronectin and collagen I. FIG. 1A. Western blotting in FIG. IB shows the effects of different siRNAs or integrin antibodies on the protein levels of FN, collagen I, a5, and P3 in CAF1299, demonstrating a5 and P3 are required for fibronectin and collagen I matrix assembly.

[0171] Images of CAF1299 cells treated with monovalent antibodies anti-avp3 (ABT10I) or anti-a5pl (ABT701), or the example bispecific antibody anti-avP3a5pl (ABT60I ; FIG. 2A) at 10 pg / mL for 72 hours were then immunostained for fibronectin and collagen I (FIG. 2B). Western blotting was also conducted showing the effects of different integrin antibodies on the protein levels of FN, collagen I, and GAPDH in CAF1299, and demonstrating antibodiestargeting integrins a5pi and avP3 significantly disrupt fibronectin and collagen I matrix assembly (FIG. 2C, FIG. 3).

[0172] Quantitative RT-PCR was conducted showing the effects of FN siRNA or different integrin antibodies on the mRNA level of FN1 and COL1A1 in cancer-associated fibroblast cell lines, CAF1299 and CAF1424 (FIG. 4). Results of RT-PCR show antibodies targeting integrins a5pi and avP3 do not reduce mRNA expression of FN1 and COL1A1.

[0173] Western blotting shows targeting integrins a5 and P3 using siRNA or antibodies reduces fibronectin matrix expression and induces soluble collagen I expression (FIG. 5). The effects of different integrin antibodies on fibronectin and collagen I in CAF1299 cells are shown in FIG. 6, with immunofluorescence staining showing the effects of different integrin antibodies on the structure and abundance of extracellular fibronectin and collagen I. Cancer- associated fibroblast cells, CAF1299, were grown for 72 hours to allow them to assemble fibronectin and collagen I. Subsequently, cells were treated with different integrin antibodies (10 pg / mL) up to 96 hours (fresh antibodies were supplemented every 48 hours), then fixed with 4% PFA and blocked with IX HBSS buffer containing 5% goat serum prior to immunofluorescence staining for fibronectin and collagen I. Antibodies targeting a5pi and avP3 are shown to disrupt pre-existing fibronectin and collagen I matrices (FIG. 6).

[0174] Cancer-associated fibroblast cells, CAF1299, were grown for 72 hours to allow them to assemble fibronectin and collagen I. Subsequently, cells were treated with different integrin antibodies (IgG4, anti-av 3 (ABT101), anti-a5pi (ABT701), or anti-avP3 / a5 l (ABT601), 10 pg / mL for all antibodies)) up to 96 hours (fresh antibodies were supplemented every 48 hours), followed by the picrosirius red staining to stain the total fiber collagen. The effects of different integrin antibodies show that antibodies targeting a5pi and avP3 can prevent the production and assembly of fibrillar collagen (FIG. 7).

[0175] Cancer-associated fibroblast cells, CAF1299, were grown for 72 hours to allow them to assemble fibronectin and collagen I. Subsequently, cells were treated with different integrin antibodies (IgG4, anti-avP3 (ABT101), anti-a5pi (ABT701), or anti-av 3 / a5 i (ABT601), 10 pg / mL for all antibodies)) up to 96 hours (fresh antibodies were supplemented every 48 hours), followed by the picrosirius red staining to stain the total fiber collagen. As shown in FIG. 8, antibodies targeting a5pi and avP3 can disrupt pre-existing fibronectin and collagen I matrices. However, antibodies targeting a5p 1 and / or avP3 do not affect fibroblast adhesion to fibronectin or collagen I matrices (FIG. 9).

[0176] Pancreatic cancer cells (human pancreatic ductal adenocarcinoma KP4 and PANCI cells were incubated with IgG4, monovalent antibodies anti-avp3 (ABT101) or anti-aS i(ABT701 ), or the example bispecific antibody anti-avP3a5pl (ABT601) at 10 ug / ml, cells fixed at final stage. FACs binding data for the three antibodies show enhanced binding of the bispecific anti-avp3 / a5pi (ABT601) at 10 pg / ml, cells fixed at final stage to pancreatic tumor cells relative to the monovalent antibodies (anti-av 3 and anti-a5 i (ABTlOl and ABT701, respectively)) (FIG. 10).

[0177] PANCI cells were incubated with antibodies (10 pg / mL): IgG4 (control), anti-uvp3 (ABTlOl), anti-a5pl (ABT701) and anti-av 3 / a5 l (ABT601), and seeded on an uncoated or fibronectin-coated plate for 24 hours, supplemented with serum-free media. The effects of different antibody treatments on the expression of YAP and its two known downstream targets, CTGF and CYR61, in pancreatic cancer cell line PANCI cells are shown in FIG. 11. These results demonstrate that YAP targets are not upregulated in tumor cells grown in the presence of antibodies targeting a5pi and avP3 (FIG. 11). In this example, YAP targets serve as a general readout for the cellular response to fibronectin in terms of biological reprogramming toward a more mesenchymal and stem-like phenotype.

[0178] Fresh lung tumor tissue was cut with a scalpel into 1-2 mm thick slices (day 0). Tissue was collected and fixed immediately to represent day 0 (dO). Two random slices from different areas of each tumor were used per condition. Slices were placed into a 24-well Transwell® plate with 1 ml DMEM complete media in the chamber and 1 ml media below. After 24 hours, slices were treated with antibody at 10 pg / ml. Media with fresh antibody was replenished every 48 hours. Samples were fixed with formalin after 72 hours (day 4, d4) of treatment. Formalin- fixed paraffin-embedded samples were stained with trichrome blue, and staining was analyzed with QuPath for % blue area. As shown in FIG. 12, the bi-specific antibody ABT601 reduced the collagen content in lung tissue samples more than ABT701 or ABTlOl, which instead increased the percent area of collagen.EXAMPLE 3: Tumor Cells Benefit from CAF-Produced Fibronectin to Overcome Isolation Stress

[0179] FN plays a critical role in ECM assembly and remodeling during a diverse range of physiological and pathological responses. To gauge the importance of CAF-produced FN during tumor initiation, KP4 human PDAC cells were injected orthotopically into the pancreas in immune-compromised nu / nu mice alone or at a 1 : 1 ratio with PD AC-derived CAFs to evaluate tumor initiation in the pancreas (FIG. 13A). After 3 weeks, bioluminescence imaging revealed bona fide tumors (luciferase signal >le+06) in 5 / 5 mice, compared to 0 / 5 mice injected with KP4 cells alone, validating the CAF-dependence of tumor initiation for thismodel. In contrast, CAFs with siRNA-mediated knockdown of FN could enable tumor initiation, a hallmark of tumor sternness, in only 1 out of 5 mice, suggesting that CAF-produced FN could largely account for the contribution of CAFs during this process. Since the siRNA- mediated knockdown efficiency begins to decline 6 days after transfection, this data suggests that CAF-produced FN is critical to enable the early steps of tumor initiation in vivo.EXAMPLE 4: CAE Interaction with Fibronectin Provides a Critical Scaffold for the Deposition of Collagen Fibers

[0180] Previous work has identified FN as a scaffold that coordinates the assembly and incorporation of additional proteins and secreted factors into the ECM (Wu et al. (2023) Nat. Cell Biol. 25(2):309-322). Indeed, CAFs isolated from PDAC patients generate a fibrotic ECM in which FN and collagen (COL) fibers clearly show a high degree of co-patteming (FIG. 13B) As evidence for FN’s role as a critical scaffold for the construction of additional matrix proteins, siRNA-mediated knockdown of fibronectin (FN1) prevents CAFs from generating collagen fibers (FIG. 13B). This reduced assembly of collagen into insoluble fibers is consistent with increased content of “soluble collagen” in conditioned media collected from CAFs with FN knockdown compared to scramble control (FIG. 13C). In contrast, knockdown of the type I collagen alpha 1 chain (COLlAl) does not affect FN fiber assembly, highlighting the unique role of FN as a critical base scaffold upon which additional ECM components such as collagen are layered. Together, these findings not only demonstrate that FN is required for CAF-mediated assembly of collagen into fibers, but the genetic approach to disrupt this process establishes proof-of-principle for target tractability.EXAMPLE 5: FN-Binding Integrins are Critical for Fibrotic Matrix Assembly

[0181] Previous studies have established that activated fibroblasts (myofibroblasts) use cell surface integrins as anchors to apply tension to fibronectin molecules, revealing cryptic sites required for polymerization into FN “fibers” that, in turn, provide a physical scaffold and repository for the assembly and recruitment of additional pro-cancer 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(l):30). Although several integrins can serve as receptors for FN, integrins avP3 and a5pi are absent on normal cells but become upregulated on a variety of 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 PD AC tumors show robust protein expression of both the a5 and P3 subunits (FIG. 14A) and cell surface expression of the avP3 and a5pi heterodimers. Similar to knockdown of FN1, collagen fibrillogenesis can be reduced by knockdown of the integrin P3 subunit (ITGB3, whose expression is the limiting factor for the formation of the avP3 heterodimer) or the integrin a5 subunit (ITGA5, whose expression similarly dictates a5pi) (FIG. 14A). Imaging permeabilized cells reveals that knockdown of FN or FN-binding integrins does not eliminate the ability of CAFs to produce collagen (as evidenced by the presence of intracellular collagen), but rather that the ability of CAFs to assemble extracellular collagen into fibers has been largely eliminated (FIG. 14A). Together, these findings indicate that FN-integrin interactions on the surface of CAFs are required for the formation of the FN fibers that function as a scaffold for other fibrotic matrix proteins, and furthermore suggest that targeting integrins a5pi / avP3 may provide an opportunity to significantly impair the contribution of CAFs to cancer progression. Since avP3 and a5pi integrins are generally absent on normal cell types but highly expressed on CAFs, targeting their function may provide an opportunity to selectively suppress the pathological fibrosis that supports PDAC progression.EXAMPLE 6: Integrin-Blocking Antibodies Prevent and Reverse CAF Assembly of ECM

[0182] To simultaneously target both integrins required for ECM production by CAFs using a single agent, we designed a novel bispecific antibody (ABT601) for dual monovalent recognition of the integrin a5pi and avP3 heterodimers (FIG. 2A). We first compared this ABT601 to its two parental control bivalent monoclonal antibodies (mAbs) that individually recognize integrins avP3 or a5pi. Commercially available antibodies recognizing integrin avP3 (LM609) and integrin a5 (P1D6) were included as additional benchmarks. Flow cytometry analysis was performed to evaluate binding of each antibody (tested at a concentration of 10 pg / mL) to two different PDAC patient-derived CAF lines, CAF-1299 and CAF-1424. For both CAF models, the median fluorescence intensity (MFI) signal for the ABT601 was slightly more than the additive sum of its two parental mAbs (FIGS. 15A-15B).

[0183] Consistent with the effects of genetic knockdown of either P3 or a5, treating CAFs with the mAbs recognizing avP3 or a5pi shows a variable ability to reduce the assembly of FN and COL fibers that may reflect the relative expression level of these integrins in a given cell population, while the ABT601 with dual recognition of integrins avP3 / a5pi produces a more complete blockade of FN and COL fibril formation than either monoclonal antibody alone(FIG. 16A). Immunostaining CAFs for FN and actin reveals punctate FN expression in the antibody-treated cells that is no longer aligned with actin fibers, confirming that this antibody does not impact the matrix adhesion and cytoskeleton of these cells. Decellularization (z.e., removal of CAFs) after 72 hours of treatment leaves behind a “CAF-ECM” substrate that clearly illustrates the significant effect of the ABT601 on both FN and COL fibers. A similar effect of the ABT601 is observed for primary hepatic stellate cells isolated from patients with liver fibrosis or primary lung fibroblasts isolated from patients with fibrotic lung disease, suggesting this antibody may have the potential for broad use as a general anti -fibrotic agent to target activated fibroblasts that depend on FN and integrins a5pi / avP3 for matrix assembly. Consistent with a change in matrix assembly but not production, treating CAFs with the ABT601 does not alter mRNA expression of either FN1 or COL1A1 genes.

[0184] To directly compare the ABT601 to a combination of monospecific mAbs in vitro, fibronectin immunostaining was evaluated for CAFs subjected to antibody treatment for 72 hours. Under these in vitro conditions for which cells are exposed to saturating antibody doses, all of the test antibodies equivalently block CAF production of FN compared to the IgG isotype control. Interestingly, while the avP3 mAb produces a significant reduction in FN levels, it does not induce the unique FN puncta as observed for the a5pi mAb, ABT601, and combination of a5pi / avP3 mAbs.

[0185] Integrins exist in a dynamic equilibrium of conformational states such that binding events are continuously being formed and dissociated. While small peptide Arg-Gly-Asp (RGD)-based competitive inhibitors cannot easily reverse integrin-ligand binding, a functionblocking pi antibody was 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 test if the ABT601 may be able to interfere with a pre-existing matrix, we allowed CAFs to deposit ECM proteins for 72 hours in culture before adding integrin-targeted antibodies. Indeed, over the span of 3 days, the ABT601 was able to disrupt pre-existing FN and COL fibers produced by CAFs (FIG. 16B), suggesting that its ability to allosterically interfere with continuously cycling on / off states of FN-integrin binding can exert a significant anti-fibrotic effect by shifting the equilibrium to a non-adhesive state.

[0186] As one biological readout for how PDAC cells respond to CAFs, we considered the ability of CAF-produced ECM to upregulate tumor cell expression of connective tissue growth factor (CTGF), a matricellular protein produced by both stromal and tumor cells that mediates their crosstalk, promotes fibrosis, and enhances 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). CAFs were cultured for 72 hours to allow for matrix deposition, then were treated for an additional 72 hours with isotype control or the ABT601. CAFs were removed to leave behind “CAF-ECM” upon which PANCI cells were plated. After an additional 24 hours, PANCI cell lysates were collected and analyzed by immunoblotting. In this model, CAF-ECM triggers a strong upregulation of CTGF in the PDAC cells, and this is prevented in the presence of the ABT601 (FIG. 16C). This assay demonstrates how blocking the two primary FN- binding integrins, avP3 and a5pi, can prevent tumor cell upregulation of CTGF in response to CAF-produced ECM.EXAMPLE 7: Disrupting FN-Binding Integrins Prevents the Ability of CAFs to Enhance Tumor Initiation

[0187] To evaluate the ability of the avP3 / a5pi ABT601 to disrupt tumor initiation in vivo, we utilized a subcutaneous xenograft model to readily detect the earliest emergence of tumor initiation over time. As for the orthotopic model (FIG. 13A), a suboptimal number of PDAC cells was injected so that initiating a tumor depended on the co-inj ection of CAFs. In this model, no palpable tumors were detected 8 weeks after subcutaneous injection of a limiting number of PANCI human PDAC cells alone, whereas co-injection of PANCI cells with CAFs at a 1 : 1 ratio produced a 100% take rate (FIG. 17A). That is, palpable tumors formed at 12 out of 12 injection sites co-injected with PANCI cells with CAFs, validating this model as a readout for CAF-dependent tumor initiation.

[0188] As observed for the orthotopic model (FIG. 13A), the “boost” in CAF-dependent tumor initiation at a subcutaneous site could be prevented using CAFs with siRNA-mediated knockdown of FN1 (si-FN), while CAFs with ITGA5 knockdown (si-a5) produced tumors at 1 out of 11 injection sites. Since the siRNA knockdowns are transient, these results suggest that the CAF contribution to tumor initiation occurs within the first several days after co-injection when tumor cells exploit CAFs to overcome isolation stress as they colonize a tumor-initiating niche. During this critical phase, eliminating CAF expression of FN or integrin a5 is sufficient to completely account for their ability to boost tumor initiation.

[0189] Accordingly, we determined whether the therapeutic ABT601 designed for dual targeting of integrins avP3 / a5pi could exert a similar activity to block tumor initiation. Since this ABT601 recognizes antigens on most species except mouse, its influence on tumor initiation in this xenograft model can be attributed to its direct binding to its antigens on the human CAFs co-injected with human PDAC cells, but not to the integrins on the surface of mouse stromal or vascular cells. Tumor cells and CAFs were premixed with 10 pg / mL ABT601immediately before injection. Once the tumor cells were injected, the antibody was then administered systemically by intraperitoneal injection twice weekly for the experiment at a dose of 10 mg / kg. Remarkably, mice treated with the avP3 / a5pi ABT601 developed palpable tumors at only 2 out of 12 injection sites (FIG. 17A), suggesting that the boost in tumor initiation offered by CAFs can be targeted therapeutically. Since the effects of the bispecific antibody are mimicked by knockdown of FN or a5 in the CAFs, blocking integrin function on CAFs has the ability to prevent tumor initiation.EXAMPLE 8: ABT601 Treatment Reduces the Fibrotic Effect of Human CAFs Co- Injected with Tumor Cells

[0190] To evaluate the mechanism(s) of action for the knockdown and blockade strategies, all palpable tumors were harvested at the 8-week endpoint of the experiment. Compared to untreated tumors, analysis of H&E-stained sections show far less stroma and but display extensive necrosis in the two tumors that formed in the ABT601 -treated group (FIG. 17B). Previous studies have documented that PDAC tumors are highly stiff due primarily to the extensive stroma and ECM deposited within the tumor, and that stiffness correlates with PDAC progression in mice and man (Cooper & Giancotti (2019) Cancer Cell 35(3):347-367; Carlson et al. (2019) Nat. Cell Biol. 21 (2):238-250). Consistent with this, mice treated with the ABT601 show significantly lower tissue stiffness throughout the entire tumor as measured by atomic force microscopy (FIG. 17C).

[0191] Analysis of the tumor microenvironment also supports the notion that the co-injection of CAFs produces a highly fibrotic and reactive tumor environment that can be targeted therapeutically. Staining tumors formed by co-injection of PANCl+CAFs using an antibody that recognizes human FN (hFN), but not mouse FN, reveals areas of intense fibrillar hFN staining (FIG. 18A), suggesting that the co-injected CAFs represent a significant producer of tumor stroma in this model relative to the host (z.e., mouse) fibroblasts. Tumor areas with dense hFN staining also show significant fibrosis (collagen polymerization), as evaluated by Masson’s trichrome (FIG. 18B) and picrosirius red staining for collagen fibers (FIG. 18C). In comparison, tumors from mice treated with the ABT601 show a complete absence of hFN, while collagen fibers appear both fewer and smaller. Since the ABT601 utilized in this study does not recognize mouse antigens, it can identify only the injected human PDAC cells and human CAFs in this xenograft model. This particularity suggests that interactions between coinjected PANCI cells and CAFs mediate the earliest steps of tumor initiation before any mouse stromal cells are recruited into the tumor microenvironment. Analysis of serial sections oftumors stained for hFN and collagen suggests that the human CAFs contribute to the production of highly fibrotic matrix, while mouse host cells that are insensitive to the ABT601 may be the source of less developed collagen networks.

[0192] In addition to the observed suppression of CAF -generated FN, we predict that in man, the ABT601 might gain additional anti -turn or efficacy by acting on activated cell types within the tumor that are known to gain expression of avP3 / a5pi, including angiogenic endothelial cells and tumor-associated macrophages. In the xenograft model, we observed a reduction in immunostaining for CD31, an endothelial cell marker used to assess tumor angiogenesis. It is therefore possible that the ABT601 may exert an indirect anti-angiogenic effect on the mouse vascular compartment (FIG. 18D), by inhibiting the accumulation of stimulatory factors and ECM proteins within the tumor microenvironment that stimulate vascular cells to generate new blood vessels (Knowles et al. (2013) Cancer Res. 73(20):6175-84; Nagpal et al (2023) Cancers (Basel) 15(4): 1216).

[0193] This work reveals that CAF-mediated assembly of FN and COL fibers is especially enabling and enhancing when PDAC cells are challenged with “isolation stress,” a state that occurs during various aspects of tumor initiation and progression. We show that disrupting the ability of CAFs to organize FN fibers results in the loss of COL fibril formation and thus provides an opportunity to interfere with intercellular communication and prevent tumor cell survival long enough to “initiate” a new tumor colony (FIG. 19).EXAMPLE 9: ABT601 Reduces Fibrotic Response in Metabolic Dysfunction-Associated Steatohepatitis (MASH) Model

[0194] A human liver spheroid model (Kim et al. (2024) STAR Protoc. 5(2): 103111) was used to evaluate the effect of ABT601 on metabolic dysfunction-associated steatohepatitis (MASH) induced fibrotic response. Primary liver cells extracted from a human donor were combined with ABT601 and allowed to form spheroids for 7 days, before being stimulated with a MASH cocktail. At day 14, spheroids and supernatant were harvested and assayed (FIG. 20A). Bright field images were taken on day 14 and spheroid diameter measured and normalized using ImageJ software (FIG. 20B). ELISA was performed on spheroid supernatant to measured Collagen I (FIG. 20C) and qPCR was performed on spheroid lysates for genes important in fibrotic response (FIG. 20D). This analysis indicated that ABT601 reduces fibrotic responses in a MASH model.Sequences:ABT601 volo cross etar h!gG4> PRO 51349_evi-5 etaracizumab.VH-(AS)-hk.LC.h4.HC.S228P.T366WHeavy Chain (SEQ ID NO:1)QVQLVESGGGWQPGRSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAKVSSGGGSTYYLDTVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHLHGSFASWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK> PRO 51350_evi-5 etaracizumab.VL-(SS)-hlv2.CHlLight Chain (SEQ ID NO:2)EIVLTQSPATLSLSPGERATLSCQASQSISNFLHWYQQRPGQAPRLLIRYRSQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSGSWPLTFGGGTKVEIKSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK> PRO 52998_evi-5 volociximab.VH-h4.HC.S228P.T363S.V366A.Y404VHeavy Chain (SEQ ID NO:3)QVQLKESGPGLVAPSQSLSITCTISGFSLTDYGVHWVRQPPGKGLEWLVVIWSDGSSTYNSALKSRMTIRKDNSKSQVFLIMNSLQTDDSAMYYCARHGTYYGMTTTGDALDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCLAKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK> PRO 52999_evi-5 volociximab.VL-hk.LCLight Chain (SEQ ID NO:4)QIVLTQSPAIMSASLGERVTMTCTASSSVSSNYLHWYQQKPGSAPNLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYLRSPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECABT701 volociximab hIgG4> PRO 53000_evi-5 volociximab.VH-h4.HC.S228PHeavy Chain (SEQ ID NO:5)QVQLKESGPGLVAPSQSLSITCTISGFSLTDYGVHWVRQPPGKGLEWLWIWSDGSSTYNSALKSRMTIRKDNSKSQVFLIMNSLQTDDSAMYYCARHGTYYGMTTTGDALDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK> PRO 52999_evi-5 volociximab.VL-hk.LCLight Chain (SEQ ID NO:6)QIVLTQSPAIMSASLGERVTMTCTASSSVSSNYLHWYQQKPGSAPNLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYLRSPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECABT101 etaracizumab hIgG4 S228P> PRO 34055_evi-5 ETAR.VH-h4.HC.S228PHeavy Chain (SEQ ID NO: 7)QVQLVESGGGWQPGRSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAKVSSGGGSTYYLDTVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHLHGSFASWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK> PRO 34054_evi-5 ETAR.VH-hk.LCLight Chain (SEQ ID NO:8)EIVLTQSPATLSLSPGERATLSCQASQSISNFLHWYQQRPGQAPRLLIRYRSQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSGSWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

CLAIMSWhat is claimed is:

1. A bispecific antibody, or antigen-binding fragment thereof, that specifically binds to two different integrins selected from integrin avP3 and integrin a5pi.

2. The bispecific antibody, or antigen-binding fragment thereof, of claim 1, comprising a first variable domain that specifically binds to integrin avP3 and / or a second variable domain that specifically binds to integrin a5pi, wherein the first variable domain and the second variable domain are different.

3. The bispecific antibody, or antigen-binding fragment thereof, of claim 2, wherein the first variable domain specifically binds to integrin avP3 and the second variable domain specifically binds to integrin a5pi.

4. The bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1- 3, further comprising an Fc domain of an IgG antibody.

5. The bispecific antibody, or antigen-binding fragment thereof, of claim 4, wherein the Fc domain is an Fc domain of an IgGl or IgG4 antibody.

6. The bispecific antibody, or antigen-binding fragment thereof, of any one of claims 3-5, wherein the first domain comprises a heavy chain VH-(AS)-hk.LC.h4.HC.S228P.T366W comprising the amino acid sequence of SEQ ID NO:1 or a sequence at least 90% identical thereto and a light chain VL-(SS)-hlv2.CHl comprising the amino acid sequence of SEQ ID NO:2 or a sequence at least 90% identical thereto.

7. The bispecific antibody, or antigen-binding fragment thereof, of any one of claims 3-6, wherein the second domain comprises a heavy chain VH-(AS)- h4.HC.S228P.T363S.V366A.Y404V comprising the amino acid sequence of SEQ ID NO:3 or a sequence at least 90% identical thereto and a light chain VL-hk.LC comprising the amino acid sequence of SEQ ID NO:4 or a sequence at least 90% identical thereto.

8. The bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1- 7, wherein the first and / or second variable domain is a humanized or human antibody domain.

9. A polynucleotide encoding the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8.

10. A vector comprising the polynucleotide of claim 9.

11. A host cell comprising the polynucleotide of claim 9 or the vector of claim 10.

12. A composition comprising the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8 and a carrier.

13. A pharmaceutical composition comprising the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, further comprising an additional therapeutic agent.

15. The pharmaceutical composition of claim 14, wherein the additional therapeutic agent is a chemotherapeutic agent.

16. A kit comprising the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8.

17. A method of preventing the binding of a cell to fibronectin, comprising contacting the cell with an effective amount of antibody or antigen-binding fragment thereof that specifically binds one or more integrins.

18. The method of claim 17, wherein the antibody is anti-a5pl (ABT701).

19. The method of claim 17, wherein the antibody or antigen-binding fragment thereof is the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8.

20. The method of claim 19, wherein the bispecific antibody, or antigen-binding fragment thereof, prevents the binding of the cell to fibrillar fibronectin.

21. The method of claim 19, wherein the bispecific antibody, or antigen-binding fragment thereof, prevents the binding of the cell to globular fibronectin.

22. The method of any one of claims 19-21, wherein the bispecific antibody, or antigenbinding fragment thereof, prevents the binding of the cell to both fibrillar and globular fibronectin.

23. The method of any one of claims 17-22, wherein the cell is a fibroblast or a mesenchymal cell.

24. The method of any one of claims 17-23, wherein the preventing the binding of fibronectin to a cell prevents collagen organization.

25. A method of treating a cancer expressing integrin avP3 and / or integrin a5pi in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, that specifically binds one or more integrins to the subject, thereby treating the cancer.

26. The method of claim 25, wherein the antibody is anti-a5pl (ABT701).

27. The method of claim 25, wherein the antibody or antigen-binding fragment thereof is the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8 or the pharmaceutical composition of any one of claims 13-15 to the subject, thereby treating the cancer.

28. A method of treating fibrotic disease comprising cells expressing integrin avP3 and / or integrin a5pi in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of antibody or antigen-binding fragment thereof that specifically binds one or more integrins, thereby treating the fibrotic disease.

29. The method of claim 28, wherein the antibody is anti-a5pi (ABT701).

30. The method of claim 28, wherein the antibody or antigen-binding fragment thereof is the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8 or the pharmaceutical composition of any one of claims 13-15, thereby treating the fibrotic disease.

31. The method of any one of claims 28-30, wherein the fibrotic disease is eye fibrosis, heart fibrosis, hepatic fibrosis, intestinal fibrosis, lung fibrosis, pancreas fibrosis, renal fibrosis, non-alcoholic steatohepatitis, systemic sclerosis, idiopathic pulmonary fibrosis, fibromyalgia, keloids or skin fibrosis.

32. The method of any one of claims 28-31, wherein the antibody, or antigen-binding fragment thereof, or pharmaceutical composition is administered to the subject intravenously, subcutaneously, or intramuscularly.

33. The method of any one of claims 28-32, wherein the cells comprise fibroblasts and / or mesenchymal cells.

34. A method of treating a cancer in a subject in need thereof, comprising the steps of: a) selecting a subject having cancer cells that are enriched for integrin avP3 and / or integrin a5pi, and optionally immune effector cells (e.g., macrophages); and b) administering a therapeutically effective amount of antibody, or antigenbinding fragment thereof, that specifically binds one or more integrins to the subject, thereby treating the cancer.

35. The method of claim 34, wherein the antibody is anti-a5pl ( ABT701).

36. The method of claim 34, wherein the antibody, or antigen-binding fragment thereof, is the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8 or the pharmaceutical composition of any one of claims 13-15 to the subject, thereby treating the cancer.

37. The method of claim 34, wherein step a) comprises obtaining a sample of the cancer from the subject and measuring the level of integrin avP3 and / or integrin a5pi, and optionally immune effector cells in the sample.

38. The method of any one of claims 34-37, wherein the cancer is an 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 multiforme), skin cancer, stomach cancer, prostate cancer, esophageal cancer, or colorectal cancer.

39. The method of any one of claims 34-38, wherein the cancer comprises a KRAS mutation or an EGFR mutation.

40. The method of any one of claims 34-39, wherein a tumor of the cancer is enriched with macrophages.

41. The method of any one of claims 34-40, wherein the cancer is an epithelial cell cancer, optionally wherein the epithelial cell cancer is a late-stage epithelial cell cancer, optionally, wherein one or more of the epithelial cells in the cancer have at least partially transitioned to mesenchymal cells.

42. The method of claim 41, wherein the epithelial cell cancer is or has become chemotherapy resistant or refractory.

43. The method of any one of claims 34-42, further comprising administering to the subject an additional cancer therapeutic agent or treatment.

44. The method of any one of claims 34-43, wherein the antibody, or antigen-binding fragment thereof, or pharmaceutical composition is administered to the subject intravenously, subcutaneously, or intramuscularly or is injected in situ into or near the cancer.

45. The method of any one of claims 25-44, wherein the subject is a human.

46. A method of inhibiting collagen organization in a subject, comprising administering to the subject a composition targeting fibronectin.

47. The method of claim 46, wherein the composition targets one or more fibronectin receptors.

48. The method of claim 47, wherein the composition targets two or more fibronectin receptors.

49. The method of claim 47 or 48, wherein the composition inhibits the expression and / or function of the one or more fibronectin receptor.

50. The method of any one of claims 47-49, wherein a fibroblast comprises the one or more fibronectin receptor.

51. The method of any one of claims 46-50, wherein the composition comprises an antisense oligonucleotide, an RNAi, or an siRNA.

52. The method of any one of claims 46-50, wherein the composition comprises an antibody, or antigen-binding fragment thereof, that specifically binds to fibronectin.

53. The method of claim 52, wherein the antibody, or antigen-binding fragment thereof, specifically binds to one or more integrins.

54. The method of claim 53, wherein the antibody or antigen-binding fragment thereof is the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8.

55. The method of claim 46, wherein the composition is the polynucleotide of claim 9.

56. A method of reversing collagen organization in a subject, comprising administering a composition targeting fibronectin.

57. The method of claim 56, wherein the composition targets one or more fibronectin receptors.

58. The method of claim 57, wherein the composition targets two or more fibronectin receptors.

59. The method of claim 57 or 58, wherein the composition inhibits the expression and / or function of the one or more fibronectin receptor.

60. The method of any one of claims 56-59, wherein the composition comprises an antisense oligonucleotide, an RNAi, or an siRNA.

61. The method of any one of claims 56-59, wherein the composition comprises an antibody, or antigen-binding fragment thereof, that specifically binds to fibronectin.

62. The method of claim 61, wherein the antibody, or antigen-binding fragment thereof, specifically binds to one or more integrins.

63. The method of claim 62, wherein the antibody, or antigen-binding fragment thereof, is the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8.

64. The method of claim 62, wherein the composition is the polynucleotide of claim 9.

65. A method of inhibiting Yes-associated protein 1 (YAP) or YAP target genes in a subject, comprising administering an antibody, or antigen-binding fragment thereof, that specifically binds one or more integrins.

66. The method of claim 65, wherein the antibody is anti-a5pi (ABT701).

67. The method of claim 65, wherein the antibody, or antigen-binding fragment thereof, is the bispecific antibody, or antigen-binding fragment thereof, of any one of claims 1-8, the polynucleotide of claim 9, or the vector of claim 10.

68. The method of claim 65, wherein the YAP target gene is CTGF or CYR61.

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