Antibody-drug conjugates for targeting fibrosis

Abstract

Antibody drug conjugates (ADCs) comprising a Lysyl Oxidase (LOX), Lysyl Oxidase homolog 1 (LOXL1), LOXL2, LOXL3 or LOXL4 inhibitor conjugated to an antibody or antigen binding fragment thereof that binds specifically to a surface marker of M2 macrophages are provided. Pharmaceutical compositions comprising the ADCs as well as methods of reducing extracellular LOX activity and treating fibrosis are also provided. Methods of producing an ADC are also provided.

Description

ANTIBODY-DRUG CONJUGATES FOR TARGETING FIBROSISCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 556,439 filed on February 22, 2024, the contents of which are all incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (TECH-P-0294-PCT.xml; Size: 32,713 bytes; and Date of Creation: February 19, 2025) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of antibody drug conjugates and fibrosis treatment.BACKGROUND OF THE INVENTION

[0004] Fibrosis, the deposition of excessive and disorganized extracellular matrix (ECM) components at the expense of normal parenchymal tissue, is a key pathological hallmark of many diseases. As a result, fibrosis affects the biochemical and biomechanical properties of the tissue, delaying its repair or regeneration, enhancing inflammation, and exacerbating disease progression. Fibrosis plays major pathological roles in a range of conditions, chronic and acute, and can affect multiple organs, including the liver, lungs, heart, kidney, and skeletal muscles, among others. It is estimated to be involved in 45% of all mortalities in the developed world. Despite its enormous clinical impact and advances in the understanding of the pathogenesis of the fibrotic process, effective therapies are limited, and the vast majority of clinical trials aiming at reducing the fibrotic burden have failed (Friedman, et al., 2013, “Therapy for fibrotic diseases: nearing the starting line”, Sci Transl Med 5, 167sr 1).

[0005] Among the various disorders impacted by fibrosis, Duchenne Muscular Dystrophy (DMD) stands out due to its severity and prevalence. DMD, caused by a mutation in the X-linked dystrophin gene, is the most common type of muscular dystrophy, affecting approximately 1 in 3,500-5,000 newborn males worldwide. Thousands of distinct mutations in dystrophin, the largest gene in the human genome spanning over 2.3 mega bases expressed in skeletal, cardiac, and smooth muscles, have been implicated in DMD. Due to the gene’s enormous size and numerous mutations, genetic treatments for DMD have remained thus far ineffective (Markati, et al., 2021, “Lessons Learned from Discontinued Clinical Developments in Duchenne Muscular Dystrophy. Front Pharmacol 12, 735912). DMD is characterized by progressive degeneration of skeletal, cardiac, and smooth muscle, leading to early mortality, usually during late adolescence to early 30s.

[0006] Muscle fibrosis, a prominent pathological feature in both DMD patients and mdx mice - the murine model of the disease, hinders tissue function, reduces blood supply and promotes an inflammatory response that further aggravates the disease. The fibrotic tissue acts as a physical barrier, preventing efficient myogenic progenitor cell migration to the muscle; creates an environment that inhibits differentiation of myogenic cells; and interferes with the successful delivery of cell-based therapies to damaged tissues. As a result, the fibrotic tissue is hardly penetrable and less responsive to tissue repair mechanisms. As fibrosis worsens and accumulates within the tissue, it accelerates disease progression, primarily due to its impact on muscle homeostasis. Identifying mechanisms to inhibit fibrosis accumulation or to enable its breakup are critically important for treating DMD as well as numerous other fibrotic diseases.

[0007] A core enzyme family implicated in the pathophysiology of diverse fibrotic conditions are the lysyl oxidases, consisting of Lysyl oxidase and Lysyl oxidase Like 1-4 (LOX, LOXL1-4). Members of this family of enzymes are critical for collagen and elastin crosslinking during ECM buildup and remodeling as well as for inducing clustering of fibronectin dimers that promote integrin activation. Dysregulated upregulation of one or more members has been observed in multiple fibrotic diseases, leading to abnormal collagen deposition and scarring. In DMD, increased LOX activity has been demonstrated in degenerating muscles leading to excessive collagen crosslinking and abnormal muscle stiffness.

[0008] It was previously demonstrated that apart from its extracellular activities within the ECM, in myogenic progenitor cells, Lox also promotes myogenesis (Yehezkely et al., 2020, “Intracellular Role for the Matrix-Modifying Enzyme Lox in Regulating Transcription Factor Subcellular Localization and Activity in Muscle Regeneration”, Developmental Cell 53, 406-417. e5). Thus, it carries dual roles - pro-myogenic and pro-fibrotic. Notably, thesetwo opposing activities are spatially separated, intracellular vs. extracellular, respectively. These findings can also possibly explain the observed short-term beneficial and long-term adverse effects in humans following treatment with beta-aminoproprionitrile (PAPN), a pan LOX inhibitor (Peacock, E.E., 1981, “Pharmacologic control of surface scarring in human beings”, Ann Surg 193, 592-597; Martin, et al., 1991, “Purkinje cell toxicity of betaaminopropionitrile in the rat”, Virchows Arch A Pathol Anat Histopathol 419, 403-408; Keiser and Sjoerdsma, 1967, “Studies on beta-aminopropionitrile in patients with scleroderma:, Clin Pharmacol Ther 8, 593-602).

[0009] Current anti-fibrotic treatments mainly involve the use of inhibitors of the TGFP pathway, which is involved in extracellular matrix secretion, or steroids to reduce the inflammatory response that is involved in fibrosis. A new anti-fibrosis therapeutic agent that can target extracellular LOX without inducing widespread toxicity is therefore greatly needed.SUMMARY OF THE INVENTION

[0010] The present invention provides antibody drug conjugates (ADCs) comprising a Lysyl Oxidase (LOX) inhibitor, Lysyl Oxidase homolog 1 (LOXL1) inhibitor, LOXL2 inhibitor, LOXL3 inhibitor or LOXL4 inhibitor conjugated to an antibody or antigen binding fragment thereof that binds specifically to a surface marker of M2 macrophages. Pharmaceutical compositions comprising the ADCs as well as methods of reducing extracellular LOX activity and treating fibrosis are also provided. Methods of producing an ADC are also provided.[Oi l] The invention is based, at least in part, on the surprising finding that weekly administration of the ADC of the invention over 3-4 months period significantly inhibited disease progression, reduced the fibrotic burden, and improved skeletal and cardiac muscle function. The results suggest that ADC treatment did not affect the balance of macrophages within the tissue, as indicated by the expression of MRC1 transcripts, the gene encoding for CD206. The ADC did stabilize myofibers, increasing their viability and decreasing necrosis, even before the onset of observable fibrosis. The absence of necrotic fibers in ADC-treated limb muscles, even after only 3 weeks of treatment, indicates beneficial activities even prior to the appearance of interstitial fibrosis, advocating for an early therapeutic window that facilitates stabilization of myofiber viability and as a result attenuation of diseaseprogression. However, late disease administration also produced benefits even in highly fibrotic tissue. The late treatment regime improved cardiac function but not at the histological level. In contrast, limb muscles and diaphragm histology showed reduced fibrosis.

[0012] According to a first aspect, there is provided an antibody drug conjugate comprising a Lysyl Oxidase (LOX) inhibitor, Lysyl Oxidase homolog 1 (LOXL1) inhibitor, LOXL2 inhibitor, LOXL3 inhibitor or LOXL4 inhibitor conjugated to an antibody or antigen binding fragment thereof that binds specifically to a surface marker of M2 macrophages.

[0013] According to some embodiments, the inhibitor is a LOX inhibitor.

[0014] According to some embodiments, the inhibitor comprises a free primary amide and wherein the inhibitor is conjugated by forming an amide bond between the free primary amide and an acidic amino acid residue of the antibody.

[0015] According to some embodiments, the inhibitor comprises a molecular weight of at most 1000 Daltons.

[0016] According to some embodiments, the inhibitor is selected from the group consisting of: CCT365623, PXS-5153A, Beta- aminopropionitrile (BAPN), aminomethylenepyridine 2 (AMT), 2-aminomethylene-5-sulfonyl-AMT, 2-aminomethylene-4-sulfonyl-AMT, Trifluoromethyl (CF3)-substituted aminomethylene-pyridine, taurine, benzylamine, isoniazid, semicarbazide and thiosemicarbazide.

[0017] According to some embodiments, the inhibitor is CCT365623.

[0018] According to some embodiments, the antibody drug conjugate comprises between 3- 10 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. According to some embodiments, the antibody drug conjugate comprises between 3-100 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof.

[0019] According to some embodiments, the antibody drug conjugate comprises 5-6 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof.

[0020] According to some embodiments, the marker of M2 macrophages is selected from CD206 and CD 163.

[0021] According to some embodiments, the marker of M2 macrophages is CD206.

[0022] According to some embodiments, the antibody is antibody 2A6A10.

[0023] According to some embodiments, the antibody comprises the same CDRs as antibody 2A6A10, comprises the same heavy chain variable region and the same light chain variable region as antibody 2A6A10 or competes with antibody 2A6A10 for binding to CD206.

[0024] According to some embodiments, the antibody is an IgG2a antibody.

[0025] According to some embodiments, the antibody drug conjugate comprises at least one molecule of CCT365623 conjugated via an amide bond to an acidic amino acid residue of the antibody.

[0026] According to another aspect, there is provided a pharmaceutical composition comprising an antibody drug conjugate of the invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0027] According to some embodiments, the pharmaceutical composition is formulated for systemic administration.

[0028] According to another aspect, there is provided a method of reducing extracellular LOX activity in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of the invention, thereby reducing extracellular LOX activity.

[0029] According to another aspect, there is provided a method of treating fibrosis in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of the invention, thereby treating fibrosis in a subject.

[0030] According to some embodiments, the fibrosis is selected from pulmonary fibrosis, liver fibrosis, renal fibrosis, peritoneal fibrosis, skeletal muscle fibrosis, skin fibrosis, pancreatic fibrosis, vascular fibrosis, cardiac fibrosis and systemic fibrosis.

[0031] According to some embodiments, the fibrosis is selected from skeletal muscle fibrosis and cardiac fibrosis.

[0032] According to some embodiments, the treating fibrosis is treating a disease selected from the group consisting of: muscular dystrophy, cystic fibrosis, systemic sclerosis, scleroderma, thyroid-associated orbitopathy, gastrointestinal cancer, inflammatory bowel disease, and Crohn’s disease.

[0033] According to some embodiments, the disease is muscular dystrophy.

[0034] According to some embodiments, the muscular dystrophy is Duchenne’s muscular dystrophy (DMD).

[0035] According to another aspect, there is provided a method of producing an antibody drug conjugate (ADC), the method comprising: a. providing an inhibitor of at least one of LOX, L0XL1, L0XL2, L0XL3 and L0XL4; b. providing an antibody or antigen binding fragment thereof that binds specifically to a surface marker of M2 macrophages; and c. conjugating the inhibitor to the antibody or antigen binding fragment thereof; thereby producing an ADC.

[0036] According to another aspect, there is provided a method of producing an antibody drug conjugate (ADC), the method comprising: a. screening agents for their ability to inhibit at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4 and selecting at least one agent that is an inhibitor of at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4; b. selecting a surface marker of M2 macrophages and producing an antibody that binds to the selected surface marker or screening antibodies or antigen binding fragments thereof for their ability to bind to the selected surface marker and selecting an antibody or antigen binding fragment thereof that binds; and c. conjugating the selected at least one agent to the produced or selected antibody or antigen binding fragment thereof; thereby producing an ADC.

[0037] According to some embodiments, step (a) comprises screening agents for their ability to inhibit LOX and selecting at least one agent that is an inhibitor of LOX.

[0038] According to some embodiments, the inhibitor comprises a free primary amide and wherein the conjugating comprises forming an amide bond between the free primary amide and an acidic amino acid residue of the antibody.

[0039] According to some embodiments, the conjugating comprises an EDC / NHS coupling reaction.

[0040] According to some embodiments, the method comprises conjugating between 3-10 molecules of the inhibitor to the antibody. According to some embodiments, the method comprises conjugating between 3-100 molecules of the inhibitor to the antibody.

[0041] According to some embodiments, the method comprises conjugating 5-6 molecules of the inhibitor to the antibody.

[0042] According to some embodiments, the method further comprises testing binding of the produced ADC to M2 macrophages and selecting an ADC that binds to M2 macrophages.

[0043] According to some embodiments, the method further comprises testing inhibition of at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4 by the ADC and selecting an ADC that inhibits at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4.

[0044] According to some embodiments, the method comprises selecting an ADC that inhibits at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4 at least as strongly as the inhibitor not conjugated to the antibody or antigen binding fragment thereof inhibits the at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4.

[0045] According to some embodiments, the marker of M2 macrophages is selected from CD206 and CD 163.

[0046] According to some embodiments, the inhibitor is selected from the group consisting of: CCT365623, PXS-5153A, Beta- aminopropionitrile (BAPN), aminomethylenepyridine 2 (AMT), 2-aminomethylene-5-sulfonyl-AMT, 2-aminomethylene-4-sulfonyl-AMT, Trifluoromethyl (CF3)-substituted aminomethylene-pyridine, taurine, benzylamine, isoniazid, semicarbazide and thiosemicarbazide.

[0047] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figures 1A-1G: Anti-CD206 specifically targets fibrotic muscles. Representative immunofluorescence images showing collagen (Coll, green) and anti-CD206 (red) staining in (1A, ID) gastrocnemius, (IB, IE) diaphragm, and (1C, IF) muscles from (left panels) 6-month-old WT C57Bl / 10ScSn and (right panels) age-matched mdx mice. DAPI marks cell nuclei (blue). In 1A-C (WT mice), collagen staining (green) shows non-fibrotic muscle tissue, and no anti-CD206 (red) staining is observed, indicating the absence of fibrosis. In contrast, in 1D-F (mdx mice), collagen staining (green) highlights fibrotic regions, and anti- CD206 staining confirms the presence of fibrosis. Yellow arrows point to fibrotic regions marked by Coll in mdx mice. Scale bar=50 um. (1G) Representative immunofluorescence images of anti-CD206 incubated with M2 macrophages for 1, 4 and 8 hours. After 1 hour of incubation, the anti-CD206 (red, grey) is mostly found on the cell membrane (marked by phalloidin, green). After 4 hours of incubation, much of the anti-CD206 is located inside the macrophages although some is still found on the cell membranes. After 8 hours of incubation, anti-CD206 is found only inside the cells. Yellow arrowheads mark membranal anti-CD206; purple arrowheads mark intracellular anti-CD206. Scale bar=5 um.

[0049] Figures 2A-2G: (2A) Schematic representation of the ADC conjugation process. (2B) Native CD206 antibodies were mixed with EDC and Sulfo-NHS reagents for 1 hour at pH 6 and 25°C. Following dialysis, CD206 mAbs were conjugated with CCT365623 inhibitor molecules for 2 hours at pH 8.4 and 25°C, followed by overnight dialysis at pH 7.4 and 4°C to yield ADC. Mass spectrometry proteomic analysis of ADC, highlighting potential modifications of CCT365623 on the amino acids glutamine and aspartic, labeled in red (i). Based on the ratio of ADC and native CD206 mAbs, peptides found only in native CD206 samples are high-lighted in red, while those showing reduced abundance are categorized into orange, yellow and green. Structural representation of ADC with highlighted potential conjugation sites (red) visualized in 360° rotation (ii). (2C) Detection efficiency percentage of mAb to human MRC1 protein by ELISA assay. (2D) Differential scanning fluorimetry analysis. The upper graph shows the fluorescence ratio (350 ng / 220 nm) as a function of temperature, while the lower graph shows the first derivative of this ratio, comparing native CD206 (black) and ADC (grey). Minor shifts in the Tm indicate changes in ADC stability upon conjugation. Results of 2C are presented as mean+standard deviation (SD) from at least three independent repetitions performed in triplicates. Results of 2D are presented as an XY graph from three independent repetitions. Statistical analysis of 2C was performed using one-way ANOVA with multiple comparisons test adjusted p-value; *p=0.0197, ****p<0.001. (2E) FACS analysis of untreated RAW264.7 cells demonstrates 1.40% of cells express CD206 (left). In contrast, following polarization, 62.6% of the cells are CD206 positive (right). (2F) Diagram of the conjugation reaction for linking CCT365623 to acid amino acid side chains. (2G) Sequences of the generic mouse IgG2A antibody heavy andlight chains. Peptides found to be conjugated to the drug (present only in the ADC and not in the control) are shown in yellow and grey and correspond to the coloring in 2B.

[0050] Figures 3A-3E: ADC targets M2 polarized macrophages while maintaining LOX inhibition. (3A-3B) Representative immunofluorescence staining of (3A) naive RAQ264.7 macrophages with (left two panels) anti-CD206 or with (right two panels) ADC, showing no binding. In contrast, immunostaining of (3B) M2 polarized macrophages shows significant membrane binding of (left two panels) anti-CD206 or (right two panels) ADC (red and grey, yellow arrowheads), demonstrating specificity toward M2 macrophages. Phalloidin marking actin is highlighted in green, and nuclei are stained in blue with DAPI. (3C) Quantitative analysis of dermal fibroblast cell area following LOX knockdown (shLOX) results in reduced cell area compared to the shControl, confirming the role of LOW in regulating cell size. (3D) Similarly, LOX inhibition with either LOX inhibitor CCT365623 or with the ADC shows a significant reduction in cell size compared to control cells. (3E) A proliferation and cell viability XTT assay performed on M2 macrophages treated with ADC, anti-CD206, or unconjugated CCT365623 shows no significant effect on cell viability across treatments. ****p<0.001. Scale bar = 10 um.

[0051] Figures 4A-4J: ADC demonstrates selective targeting of fibrotic regions and macrophages in dystrophic muscle. (4A-F) Immunofluorescence analysis of Coll (green) and ADC (red) distribution in (4A, 4D) gastrocnemius, (4B, 4E) diaphragm and (4C, 4F) heart muscles from 6-month-old (4A-C) WT C57Bl / 10ScSn and (4D-4F) mdx mice. ADC accumulation is undetectable in non-fibrotic WT tissues (4A-4C, right panels) but exhibits specific localization to Coll-enriched regions in mdx muscles (4D-4F, right panels, yellow arrowheads). Nuclei are visualized with DAPI (blue). (4G-4I) Immuno staining of ADC colocalization in mdx gastrocnemius sections labeled with WGA (green) and DAPI (blue). (4G) ADC (red) shows no colocalization with PDGFRa+ expressing FAPs (magenta). (4H) ADC (red) displays no overlap with CD31+ endothelial cells (yellow). (41) ADC (red) shows selective association with F4 / 80-expressing macrophages (cyan). Yellow arrowheads denote representative cells in higher magnification insets. Scale bar = 50 um. (4J) Representative immunofluorescence images from ADC in WT C57Bl / 10ScSn and mdx mice of non-muscle, non-fibrotic kidneys, livers and lungs. No staining of the ADC is observed in the WT or in the mdx mice. E-cadherin (green), ADC (red, grey) and nuclei stained with DAPI (blue). Scale bar = 200 um.

[0052] Figures 5A-5F: Long-term weekly ADC treatments significantly improve muscle function in mdx mice. (5A, 5D) Treadmill results over a three-months period (3-6months of age). ADC-treated mdx mice demonstrated significantly improved running capabilities compared to the unconjugated anti-CD206 or unconjugated CCT365623 groups. Significant improvements were observed as early as the fourth week of treatment. (5B-5C, 5E-5F) Cardiac function, as assessed by echocardiography after, was further significantly improved in the ADC-treated mice. (5B, 5E) Monthly assessments of fractional shortening and (5C, 5F) ejection fraction demonstrate that mice treated with ADC exhibited enhanced cardiac function. ADC (20 ug / 100 ul; N=6), unconjugated CCT365623 (16.3 ug / 100 ul per mouse; N=5), unconjugated anti-CD206 (20 ug / 100 ul; N=5), WT (untreated N=5). *** pval<0.005. 5D-5F show additional controls.

[0053] Figures 6A-6G: Long-term weekly ADC treatments significantly attenuate fibrosis in mdx mice. (6A-6D) Representative images of Coll immunostaining in (6A-6B) hearts and (6C-6D) diaphragms of mdx mice. Mice were treated with weekly IP injections of (6A, 6C) IgG or (6B, 6D) ADC. (6E-6F) Boxplot quantification of Coll immuno staining shows significantly reduced Coll amount in 6 months old ADC-treated mdx mouse (6E) hearts and (6F) diaphragms compared to IgG controls. ADC (20 ug / 100 ul; N=6), IgG (20 ug / 100 ul; N=5). ***pval<0.005, **pval<0.01. Scale bar = 80 um. (6G) RT-PCR results depicting expression of the fibrotic genes TGFB 1, Collal, fibronectin (FN), Lox, and MRC1 (CD206) in diaphragms and cardiac muscles of 3-6 months treated mice (N=6 of each). ns=not significant, *pval<0.05, **pval<0.01, ***<0.005, ****pval<0.001.

[0054] Figures 7A-7K: ADC inhibits myofiber necrosis. (7A-7C) Representative immunofluorescence images of gastrocnemius muscle for laminin (red), and myofiber necrosis (green) in (7A) untreated, (7B) IgG- and (7C) ADC-treated mdx mice. (7D) Boxplot quantification of the percentage of necrotic muscle fibers per image at 6 months. (7E-7G) Regenerative fibers marked by embryonic myosin heavy chain (MYH3, green) and laminin (red) staining in (7E) untreated, (7F) IgG- and (7G) ADC-treated mdx mice. (7H) Boxplot quantification of regenerative fibers per image at 6 months. (71-7 J) Representative immunofluorescence images of gastrocnemius muscle sections from (71) IgG-treated and (7 J) ADC-treated mice showing necrotic fibers treated for only three weeks. Left panels show laminin staining (red), IgG-positive necrotic fibers (green) and DAPI nuclear staining (blue). Corresponding single change images (right panels) show IgG-positive necrotic fibers alone. ADC (20 ug / 100 ul; N=6), IgG (20 ug / 100 ul; N=6), Untreated (N=8), *pval,0.05, ***pval<0.005, ****pval<0.001. Scale bars = 80 um for 7A-7G and 50 um for 7I-7J. (7K) Histograms of cross-section area of myofibers in the gastrocnemius and diaphragm muscles.No significant differences between the treatment regimens are observed. IgG (N=6 mice) and ADC (N=6 mice).

[0055] Figures 8A-8I: Long-term weekly ADC treatments enhance heart function and reduce fibrosis in late disease stages. (8A) Treadmill results over a four-month period (6- 10 months of age). No significant differences were observed between the ADC-treated and the distinct control mdx mice; their exercise performance was significantly weaker than WT mice. (8B-8C) Cardiac function, as assessed by echocardiography, was significantly improved in the ADC-treated mdx mice. Monthly assessment of (8B) fractional shortening and (8C) ejection fraction demonstrate that mice treated with ADC exhibited enhanced cardiac function. (8D, 8F, 8H) Representative Masson trichrome staining of (8D) diaphragm, (8F) heart and (8H) gastrocnemius muscles. (8E, 8G, 81) Boxplot quantification of percentage fibrosis in the (8E) diaphragm, (8G) heart and (81) gastrocnemius muscles.DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention, in some embodiments, provides antibody drug conjugates (ADCs) comprising a Lysyl Oxidase (LOX), Lysyl Oxidase homolog 1 (LOXL1), LOXL2, LOXL3 or LOXL4 inhibitor conjugated to an antibody or antigen binding fragment thereof that binds specifically to a surface marker of M2 macrophages. Pharmaceutical compositions comprising the ADCs are also provided. Methods of reducing extracellular LOX activity and methods of treating fibrosis by administering the ADCs to a subject are also provided. Additionally, methods of producing an ADC are also provided.

[0057] The invention is based, at least in part, on the hypothesized that targeting LOX inhibition primarily and specifically to the ECM of fibrotic tissues, without affecting its intracellular activities, will inhibit fibrosis accumulation while facilitating muscle regeneration. Fibroadipogenic progenitors (FAPs) are key players in the fibrotic reaction and Lysyl oxidases are known to be expressed and secreted by these cells. However, FAPs as do the Lysyl oxidases, also participate in homeostatic processes in multiple tissues and therefore targeting LOX inhibition to these FAPs could entail undesired effects.

[0058] To overcome this, herein an antibody-drug conjugate (ADC) was produced in which a LOX inhibitor, CCT365623, was conjugated to an antibody targeting CD206, encoded by the mannose receptor C-type 1 (MRC1) gene. It is generally shown that LOX and LOX homolog inhibitors, which are generally toxic when administered systemically, can betargeted to areas of fibrosis by conjugation to an anti-M2 macrophage antibody. CD206 (or CD163) is expressed on M2 macrophages highly enriched in fibrotic regions, including those observed in DMD. CD206 on M2 macrophages is an optimal target because these macrophages are abundant in fibrotic tissues and play a crucial role in fibrosis. Notably, transcriptomic analysis of myogenic cells demonstrates it is not expressed in the latter. This ADC combines the specificity of an antibody with the potency of a small molecule drug, thus specifically targeting the LOX enzyme responsible for fibrosis. It is demonstrated that this ADC specifically localizes to fibrotic regions but not to healthy tissues even in diseased mdx mice. It was further found that weekly administration of the ADC over long periods of 3-4 months promotes cardiac and skeletal muscle function, inhibits myofiber necrosis and reduces muscle fibrosis in mdx mice. Altogether, these observations suggest this ADC can serve as a novel route for treating multiple LOX-dependent fibrotic diseases such as DMD, promoting tissue healing and elongating the window of time for administering other diseasemodifying drugs. This is achieved specifically by binding a surface marker so that the inhibitors have access to the extracellular milieu where LOX and LOX homologs are active but cannot inhibit intracellular LOX activity which is the source of the reported toxicity.

[0059] By a first aspect, there is provided an antibody drug conjugate (ADC) comprising a Lysyl Oxidase (LOX) inhibitor, Lysyl Oxidase homolog 1 (LOXL1) inhibitor, LOXL2 inhibitor, LOXL3 inhibitor or LOXL4 inhibitor conjugated to an antibody or antigen binding fragment thereof.

[0060] By another aspect, there is provided an antibody drug conjugate (ADC) comprising an inhibitor of lysyl oxidase activity conjugated to an antibody or antigen binding fragment thereof.

[0061] As used herein, the term “antibody drug conjugate (ADC)” refers to a complex molecule comprising an antibody or antigen binding fragment thereof linked to a biologically active payload or drug. In some embodiments, the ADC is a composition. In some embodiments, the ADC is therapeutic. In some embodiments, the ADC is a drug. In some embodiments, the antibody or antigen binding fragment thereof and the drug are linked. In some embodiments, the linking is by a linker. In some embodiments, the ADC is not a fusion protein. In some embodiments, linked is conjugated. In some embodiments, conjugated in linked.

[0062] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide chainshaving three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form an antibody binding site. An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelised, CDR-grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled in soluble or bound form as well as fragments, including epitope-binding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences. An antibody may be from any species. The term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab', F(ab')2 single stranded antibody (svFC), dimeric variable region (Diabody) and disulphide-linked variable region (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Antibody fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. The skilled artisan will further appreciate that other fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH)~ Fc fusions and scFv-scFv-Fc fusions.

[0063] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. In some embodiments, the antibody or antigen binding fragment thereof is an antibody. In some embodiments, the antibody is a fully antibody comprising two heavy chains and two light chains. In some embodiments, the antibody or antigen binding fragment thereof is a single chain antibody. In some embodiments, the antibody or antigen binding fragment thereof a mouse antibody. In some embodiments, the antibody or antigen binding fragment thereof is a humanized antibody. In some embodiments, the antibody or antigen binding fragment thereof is a human antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the IgG2 antibody is an IgG2A antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody does not induce antibody dependent cell cytotoxicity (ADCC). In some embodiments, the antibody does not induce complement dependent cytotoxicity (CDC).

[0064] In some embodiments, the antibody comprises a heavy chain constant region. In some embodiments, the heavy chain constant region comprises AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAP NLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT QTHREDYNSTLRVVSALPIQHQWMSGKEFKCKVNNKDLAPAPIERTISKPKGSVR APQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVL DSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR (SEQ ID NO: 34). In some embodiments, the heavy chain constant region consists of SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 92, 95, 97, or 99% identity to SEQ ID NO: 34. Each possibility represents a separate embodiment of the invention. In some embodiments, the heavy chain constant region comprises a sequence with at least 85% identity to SEQ ID NO:34. In some embodiments, the heavy chain constant region comprises a sequence with at least 95% identity to SEQ ID NO: 34. In some embodiments, the heavy chain constant region comprises a sequence with at least 99% identity to SEQ ID NO: 34. In some embodiments, the heavy chain constant region consists of a sequence with at least 85% identity to SEQ ID NO: 34. In some embodiments, the heavy chain constant region consists of a sequence with at least 95% identity to SEQ ID NO: 34. In some embodiments, the heavy chain constant region consists of a sequence with at least 99% identity to SEQ ID NO: 34.

[0065] In some embodiments, the antibody comprises a light chain constant region. In some embodiments, the light chain constant region comprises RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSW TDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 35). In some embodiments, the light chain constant region consists of SEQ ID NO: 35. In some embodiments, the light chain constant region comprises or consists of a sequence with at least 70, 75, 80, 85, 90, 92, 95, 97, or 99% identity to SEQ ID NO: 35. Each possibility represents a separate embodiment of the invention. In some embodiments, the light chain constant region comprises a sequence with at least 85% identity to SEQ ID NO:35. In some embodiments, the light chain constant region comprises a sequence with at least 95% identity to SEQ ID NO: 35. In some embodiments, the light chain constant region comprises a sequence with at least 99% identity to SEQ ID NO: 35. In some embodiments, the light chain constant region consists of a sequence with at least 85% identity to SEQ ID NO: 35. In some embodiments, the light chain constant region consists of a sequence with at least 95% identity to SEQ ID NO: 35. In some embodiments, the light chain constant region consists of a sequence with at least 99% identity to SEQ ID NO: 35.

[0066] In some embodiments, the antibody or antigen binding fragment thereof binds to M2 macrophages. In some embodiments, the antibody or antigen binding fragment thereof is specific to M2 macrophages. In some embodiments, binds to is specifically binds to. In some embodiments, the antibody or antigen binding fragment thereof binds to a surface protein on M2 macrophages. In some embodiments, the surface protein is a marker for M2 macrophages. In some embodiments, the surface protein is specific to M2 macrophages. In some embodiments, the surface protein is more highly expressed on M2 macrophages than all other cell types. In some embodiments, all other cell types are all other healthy cell types. In some embodiments, more highly is by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000% greater expression. Each possibility represents a separate embodiment of the invention.

[0067] In some embodiments, the surface protein is a receptor. In some embodiments, the surface protein is CD206. In some embodiments, the surface protein is CD 163. In some embodiments, the surface protein is selected from the group consisting of CD206 and CD163. In some embodiments, the CD206 is mammalian. In some embodiments, the CD163 is mammalian. In some embodiments, the mammal is human.

[0068] CD206 is also known as Mannose receptor, Macrophage mannose receptor and mannose receptor, C type 1 (MRC1). CD206 is encoded by the mrcl gene and the human gene is available at Entrez gene ID 4360. The human CD206 protein sequence is available at Uniprot ID P22897. The human CD206 mRNA is provided in RefSeq NM_002438 and the human CD206 protein is provided in RefSeq NP_002429. In some embodiments, CD206 comprises the amino acid sequenceMRLPLLLVFASVIPGAVLLLDTRQFLIYNEDHKRCVDAVSPSAVQTAACNQDAES QKFRWVSESQIMSVAFKLCLGVPSKTDWVAITLYACDSKSEFQKWECKNDTLLGI KGEDLFFNYGNRQEKNIMLYKGSGLWSRWKIYGTTDNLCSRGYEAMYTLLGNAN GATCAFPFKFENKWYADCTSAGRSDGWLWCGTTTDYDTDKLFGYCPLKFEGSES LWNKDPLTSVSYQINSKSALTWHQARKSCQQQNAELLSITEIHEQTYLTGLTSSLT SGLWIGLNSLSFNSGWQWSDRSPFRYLNWLPGSPSAEPGKSCVSLNPGKNAKWEN LECVQKLGYICKKGNTTLNSFVIPSESDVPTHCPSQWWPYAGHCYKIHRDEKKIQR DALTTCRKEGGDLTSIHTIEELDFIISQLGYEPNDELWIGLNDIKIQMYFEWSDGTPV TFTKWLRGEPSHENNRQEDCVVMKGKDGYWADRGCEWPLGYICKMKSRSQGPE IVEVEKGCRKGWKKHHFYCYMIGHTLSTFAEANQTCNNENAYLTTIEDRYEQAFL TSFVGLRPEKYFWTGLSDIQTKGTFQWTIEEEVRFTHWNSDMPGRKPGCVAMRTGIAGGLWDVLKCDEKAKFVCKHWAEGVTHPPKPTTTPEPKCPEDWGASSRTSLCFK LYAKGKHEKKTWFESRDFCRALGGDLASINNKEEQQTIWRLITASGSYHKLFWLG LTYGSPSEGFTWSDGSPVSYENWAYGEPNNYQNVEYCGELKGDPTMSWNDINCE HLNNWICQIQKGQTPKPEPTPAPQDNPPVTEDGWVIYKDYQYYFSKEKETMDNAR AFCKRNFGDLVSIQSESEKKFLWKYVNRNDAQSAYFIGLLISLDKKFAWMDGSKV DYVSWATGEPNFANEDENCVTMYSNSGFWNDINCGYPNAFICQRHNSSINATTV MPTMPSVPSGCKEGWNFYSNKCFKIFGFMEEERKNWQEARKACIGFGGNLVSIQN EKEQAFLTYHMKDSTFSAWTGLNDVNSEHTFLWTDGRGVHYTNWGKGYPGGRR SSLSYEDADCVVIIGGASNEAGKWMDDTCDSKRGYICQTRSDPSLTNPPATIQTDG FVKYGKSSYSLMRQKFQWHEAETYCKLHNSLIASILDPYSNAFAWLQMETSNERV WIALNSNLTDNQYTWTDKWRVRYTNWAADEPKLKSACVYLDLDGYWKTAHCN ESFYFLCKRSDEIPATEPPQLPGRCPESDHTAWIPFHGHCYYIESSYTRNWGQASLE CLRMGSSLVSIESAAESSFLSYRVEPLKSKTNFWIGLFRNVEGTWLWINNSPVSFVN WNTGDPSGERNDCVALHASSGFWSNIHCSSYKGYICKRPKIIDAKPTHELLTTKAD TRKMDPSKPSSNVAGVVIIVILLILTGAGLAAYFFYKKRRVHLPQEGAFENTLYFNS QSSPGTSDMKDLVGNIEQNEHSVI (SEQ ID NO: 25). In some embodiments, CD206 consists of SEQ ID NO: 25. Antibodies to CD206 are well known in the art and any such antibody may be used. Examples of such antibodies can be found in Table 1. A sufficient number of anti-CD206 antibodies are known in the art such that a skilled artisan would be in possession of the genus of anti-CD206 antibodies. In some embodiments, the anti-CD206 antibody is selected from the antibodies provided in Table 1. In some embodiments, the anti- CD206 antibody is an antibody provided in Table 1.

[0069] Table 1: Anti-CD206 antibodies

[0070] CD163 is also known as Scavenger receptor cysteine-rich type 1 protein M130, MM130 and SCARI1. CD163 is encoded by the CD163 gene and the human gene is available at Entrez gene ID 9332. The human CD163 protein sequence is available at Uniprot ID Q86VB7. The human CD 163 mRNA is provided in RefSeq NM_004244, NM_203416, NM_001370145 and NM_001370146 and the human CD163 protein is provided in RefSeq NP_004235, NP_981961, NP_001357074 and NP_001357075. In some embodiments, CD 163 comprises the amino acid sequenceMSKLRMVLLEDSGSADFRRHFVNLSPFTITVVLLLSACFVTSSLGGTDKELRLVDG ENKCSGRVEVKVQEEWGTVCNNGWSMEAVSVICNQEGCPTAIKAPGWANSSAGS GRIWMDHVSCRGNESALWDCKHDGWGKHSNCTHQQDAGVTCSDGSNLEMRLT RGGNMCSGRIEIKFQGRWGTVCDDNFNIDHASVICRQLECGSAVSFSGSSNFGEGS GPIWFDDLICNGNESALWNCKHQGWGKHNCDHAEDAGVICSKGADLSLRLVDG VTECSGRLEVRFQGEWGTICDDGWDSYDAAVACKQLGCPTAVTAIGRVNASKGF GHIWLDSVSCQGHEPAIWQCKHHEWGKHYCNHNEDAGVTCSDGSDLELRLRGG GSRCAGTVEVEIQRLLGKVCDRGWGLKEADVVCRQLGCGSALKTSYQVYSKIQA TNTWLFLSSCNGNETSLWDCKNWQWGGLTCDHYEEAKITCSAHREPRLVGGDIP CSGRVEVKHGDTWGSICDSDFSLEAASVLCRELQCGTVVSILGGAHFGEGNGQIW AEEFQCEGHESHLSLCPVAPRPEGTCSHSRDVGVVCSRYTEIRLVNGKTPCEGRVE LKTLGAWGSLCNSHWDIEDAHVLCQQLKCGVALSTPGGARFGKGNGQIWRHMF HCTGTEQHMGDCPVTALGASLCPSEQVASVICSGNQSQTLSSCNSSSLGPTRPTIPE ESAVACIESGQLRLVNGGGRCAGRVEIYHEGSWGTICDDSWDLSDAHVVCRQLG CGEAINATGSAHFGEGTGPIWLDEMKCNGKESRIWQCHSHGWGQQNCRHKEDA GVICSEFMSLRLTSEASREACAGRLEVFYNGAWGTVGKSSMSETTVGVVCRQLGC ADKGKINPASLDKAMSIPMWVDNVQCPKGPDTLWQCPSSPWEKRLASPSEETWIT CDNKIRLQEGPTSCSGRVEIWHGGSWGTVCDDSWDLDDAQVVCQQLGCGPALK AFKEAEFGQGTGPIWLNEVKCKGNESSLWDCPARRWGHSECGHKEDAAVNCTDI SVQKTPQKATTGRSSRQSSFIAVGILGVVLLAIFVALFFLTKKRRQRQRLAVSSRGE NLVHQIQYREMNSCLNADDLDLMNSSENSHESADFSAAELISVSKFLPISGMEKEA ILSHTEKENGNL (SEQ ID NO: 26). In some embodiments, CD 163 consists of SEQ ID NO: 26. Antibodies to CD163 are well known in the art and any such antibody may be used. Examples of such antibodies can be found in Table 2. A sufficient number of anti-CD163 antibodies are known in the art such that a skilled artisan would be in possession of the genusof anti-CD163 antibodies. In some embodiments, the anti-CD163 antibody is selected from the antibodies provided in Table 2. In some embodiments, the anti-CD163 antibody is an antibody provided in Table 2.

[0071] Table 2: Anti-CD163 antibodies

[0072] In some embodiments, the antibody is the 2A6A10 antibody. In some embodiments, the anti-CD206 antibody is the 2A6A10 antibody. The 2A6A10 antibody is commercially available and can be purchased for example from Thermo Fisher Scientific (thermofisher.com / antibody / product / CD206-Antibody-clone-2A6A10-Monoclonal / 60143- 1-IG) and Proteintech (ptglab.com / products / CD206-Antibody-CL488-60143.htm). In some embodiments, the antibody or antigen binding fragment thereof comprises the same six CDRs as the 2A6A10 antibody. In some embodiments, the six CDRs are three heavy chain CDRs and three light chain CDRs. In some embodiments, the antibody or antigen binding fragment thereof comprises the same heavy chain variable region as the 2A6A10 antibody. In some embodiments, the antibody or antigen binding fragment thereof comprises the same heavy chain variable region as the 2A6A10 antibody and the same three light chain CDRs as the 2A6A10 antibody. In some embodiments, the antibody or antigen binding fragment thereof comprises the same light chain variable region as the 2A6A10 antibody. In some embodiments, the antibody or antigen binding fragment thereof comprises the same light chain variable region as the 2A6A10 antibody and the same three heavy chain CDRs as the 2A6A10 antibody. In some embodiments, the antibody or antigen binding fragment thereof comprises the same light chain variable region and the same heavy chain variable region as the 2A6A10 antibody. In some embodiments, the antibody or antigen binding fragmentthereof comprises the same heavy chain as the 2A6A10 antibody. In some embodiments, the antibody or antigen binding fragment thereof comprises the same light chain as the 2A6A10 antibody. In some embodiments, the antibody or antigen binding fragment thereof comprises the same heavy chain and the same light chain as the 2A6A10 antibody. In some embodiments, the antibody or antigen binding fragment thereof competes with the 2A6A10 antibody for binding to CD206. In some embodiments, the antibody or antigen binding fragment thereof binds to the same epitope of CD206 as the 2A6A10 antibody.

[0073] While there is a protein named Lysyl Oxidase (LOX), it will be understood that as used herein the term “lysyl oxidase” activity is not limited to the activity of LOX but rather refers to the activity produced by all proteins encoded by genes of the lysyl oxidase gene family or all family members of the lysyl oxidase enzymatic family. This gene and enzymatic family contains five members: LOX, LOXL1, LOXL2, LOXL3 and LOXL4.

[0074] In some embodiments, the inhibitor is a LOX inhibitor. In some embodiments, the inhibitor is a LOXL1 inhibitor. In some embodiments, the inhibitor is a LOXL2 inhibitor. In some embodiments, the inhibitor is a LOXL3 inhibitor. In some embodiments, the inhibitor is a LOXL4 inhibitor. In some embodiments, the inhibitor is a pan LOX inhibitor. In some embodiments, the inhibitor is a dual inhibitor. In some embodiments, the dual inhibitor is a LOX / LOXL2 inhibitor. In some embodiments, the dual inhibitor inhibits at least two of LOX, LOXL1, LOXL2, LOXL3 and LOXL4. In some embodiments, the dual inhibitor is a LOXL2 / LOXL3 inhibitor. In some embodiments, the inhibitor is a specific inhibitor. In some embodiments, the inhibitor is specific to LOX. In some embodiments, the inhibitor is specific to LOXL2. In some embodiments, the inhibitor is specific to LOX and LOXL2.

[0075] In some embodiments, the inhibitor is a small molecule inhibitor. In some embodiments, the inhibitor is not a protein. In some embodiments, the inhibitor is an organic compound. In some embodiments, the inhibitor comprises a molecular weight of less than 10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2500, 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, or 400 Daltons. Each possibility represents a separate embodiment of the invention. In some embodiments, the inhibitor comprises a molecular weight of less than 1000 Daltons. In some embodiments, the inhibitor comprises a molecular weight of less than 500 Daltons. In some embodiments, the inhibitor comprises a molecular weight of less than 450 Daltons. In some embodiments, the inhibitor comprises a molecular weight of at most 10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2500, 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, or 400 Daltons. Each possibility represents a separate embodiment of the invention. In some embodiments, the inhibitorcomprises a molecular weight of at most 1000 Daltons. In some embodiments, the inhibitor comprises a molecular weight of at most 500 Daltons. In some embodiments, the inhibitor comprises a molecular weight of at most 450 Daltons.

[0076] In some embodiments, the inhibitor comprises a primary amide. A primary amide is an NH2 in which the nitrogen is covalently bonded to one other atom in addition to the two hydrogens. In some embodiments, the inhibitor comprises a free primary amide. In some embodiments, the primary amide is reacted with the antibody to produce the conjugate.

[0077] In some embodiments, the inhibitor is selected from the group consisting of: CCT365623, PXS-5153A, Beta-aminopropionitrile (BAPN), aminomethylenepyridine 2 (AMT), 2-aminomethylene-5-sulfonyl-AMT, 2-aminomethylene-4-sulfonyl-AMT, Trifluoromethyl (CF3)-substituted aminomethylene-pyridine, taurine, benzylamine, allylamine, isoniazid, semicarbazide and thiosemicarbazide. In some embodiments, the inhibitor is selected from the group consisting of: CCT365623, PXS-5153A, Beta- aminopropionitrile (BAPN), aminomethylenepyridine 2 (AMT), 2-aminomethylene-5- sulfonyl-AMT, 2-aminomethylene-4-sulfonyl-AMT, Trifluoromethyl (CF3)-substituted aminomethylene-pyridine, taurine, isoniazid, semicarbazide and thiosemicarbazide. In some embodiments, the inhibitor is a benzylamine. In some embodiments, the inhibitor is an allylamine. In some embodiments, the inhibitor is selected from the group consisting of: CCT365623, PXS-5153A, Beta-aminopropionitrile (BAPN), aminomethylenepyridine 2 (AMT), 2-aminomethylene-5-sulfonyl-AMT, 2-aminomethylene-4-sulfonyl-AMT, taurine, benzylamine, allylamine, isoniazid, semicarbazide and thiosemicarbazide, all of which contain a primary amide. In some embodiments, the inhibitor is selected from BAPN and CCT365623. Further LOX inhibitors are also disclosed in International Patent Applications W02017141049, W02019073251, W02020099886, and W02019234418, the contents of which are hereby incorporated by reference in their entirety. Any of these LOX inhibitors may also be used as part of the conjugate of the invention.

[0078] In some embodiments, the inhibitor is CCT365623. CCT365623 is identified by CAS number 2126136-98-7. CCT365623 is also identified by the formula CtsH isCINCLSs. It has a molecular weight of 443.99 Daltons. It can also be written in SMILES notation as: NCC 1=CC=C(S(=O)(C2=CC(C3=CC=CC=C3 )=CC(S(=O)(C)=O)=C2)=O)S 1.Cl. Finally, CCT365623 is depicted in Figure 2F. As shown in Figure 2F, CCT365623 contains a free primary amide that can be reacted with either acidic amino acid side chain (glutamic acid or aspartic acid) to covalently conjugate the inhibitor to the antibody via an amide bond. CCT365623 is disclosed in International Patent Applications W02017141049 as are otherLOX inhibitors with structures similar to CCT365623. Any such inhibitors and molecules with a similar core (i.e., derivatives of CCT365623) may be used as part of the conjugate of the invention. In some embodiments, the inhibitor is CCT365623 or a derivative thereof. In some embodiments, the derivative of CCT365623 is a molecule discloses in International Patent Applications W02017141049. In some embodiments, CCT365623 has the formula

[0079] In some embodiments, the inhibitor is BAPN. In some embodiments, the inhibitor is PXS-5153A. In some embodiments, the inhibitor is AMT. In some embodiments, AMT is aminomethyl pyridine 2. In some embodiments, the inhibitor is 2-aminomethylene-5- sulfonyl-AMT. In some embodiments, the inhibitor is 2-aminomethylene-4-sulfonyl-AMT. In some embodiments, the inhibitor is Trifluoromethyl (CF3)-substituted aminomethylenepyridine. In some embodiments, the inhibitor is taurine. In some embodiments, the inhibitor is benzylamine. In some embodiments, the inhibitor is allylamine. In some embodiments, the inhibitor is isoniazid. In some embodiments, the inhibitor is semicarbazide. In some embodiments, the inhibitor is thiosemicarbazide.

[0080] In some embodiments, the conjugation is by a covalent bond. In some embodiments, the linking is covalent linking. In some embodiments, the linking is by an amide bond. In some embodiments, the amide bond is formed between a primary amide of the inhibitor and the antibody. In some embodiments, the bond is between a primary amide of the inhibitor and an acidic amino acid residue of the antibody. In some embodiments, the conjugation is an amide bond between the primary amide of the inhibitor and an acidic amino acid residue of the antibody. In some embodiments, the acidic amino acid residue is selected from an aspartic acid (D) residue and a glutamic acid (E) residue.

[0081] In some embodiments, the ADC comprises a plurality of molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the ADC comprises between 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-25, 1-20, 1-19,1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2- 100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-25, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14,2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50,3-40, 3-30, 3-25, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8,3-7, 3-6, 3-5, 3-4, 4-100, 4-90, 4-80, 4-70, 4-60, 4-50, 4-40, 4-30, 4-25, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-25, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5- 10, 5-9, 5-8, 5-7, 5-6, 6-100, 6-90, 6-80, 6-70, 6-60, 6-50, 6-40, 6-30, 6-25, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-100, 7-90, 7-80, 7-70, 7- 60, 7-50, 7-40, 7-30, 7-25, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, or 7-8 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the ADC comprises between 3-10 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the ADC comprises between 3-100 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the ADC comprises between 1-10 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the ADC comprises between 1-100 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the ADC comprises between 5-6 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof.

[0082] In some embodiments, the ADC comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 95 or 100 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the ADC comprises about 5 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the ADC comprises about 6 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof.

[0083] In some embodiments, the ADC comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the ADC comprises at least 5 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the ADC comprises at least 6 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof.

[0084] In some embodiments, the ADC comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 100 molecules of the inhibitorconjugated to the antibody or antigen binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the ADC comprises at most 100 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the ADC comprises at most 10 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the ADC comprises at most 6 molecules of the inhibitor conjugated to the antibody or antigen binding fragment thereof.

[0085] In some embodiments, the inhibitor is conjugated to the heavy chain of the antibody. In some embodiments, the inhibitor is conjugated to the light chain of the antibody. In some embodiments, the inhibitor is conjugated to the heavy chain and to the light chain of the antibody. In some embodiments, the inhibitor is conjugated to the constant region of the heavy chain of the antibody. In some embodiments, the inhibitor is conjugated to the constant region of the light chain of the antibody. In some embodiments, the inhibitor is conjugated to the constant region of the heavy chain and to the constant region of the light chain of the antibody. In some embodiments, the light chain is a kappa light chain. In some embodiments, the light chain is a lambda chain.

[0086] In some embodiments, the inhibitor is conjugated to E430 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to E357 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to E356 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to E / D356 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to D356 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to E355 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to E388 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to E395 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to D399 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to D401 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to D134 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to DI 35 of an IgG heavy chain. In some embodiments, the inhibitor IS conjugated to E332 of an IgG heavy chain. In some embodiments, the inhibitor IS conjugated to E333 of an IgG heavy chain. In some embodiments, the inhibitor IS conjugated to D326 of an IgG heavy chain. In some embodiments, the inhibitor IS conjugated to D327 of an IgG heavy chain. In some embodiments, the inhibitor IS conjugated to D211 of an IgG heavy chain. In some embodiments, the inhibitor IS conjugated to D212 of an IgG heavy chain. In someembodiments, the inhibitor is conjugated to E215 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to E216 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to E89 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to E90 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to D90 of an IgG heavy chain. In some embodiments, the inhibitor is conjugated to D91 of an IgG heavy chain. In some embodiments, the IgG is IgG2. In some embodiments, the numbering is according to the EU index. In some embodiments, the numbering is as defined by the EU index. In some embodiments, the EU index is as defined by Kabat. In some embodiments, the numbering is with respect to IgG2. In some embodiments, the numbering is with respect to a mouse antibody. The conversion of positions in a mouse antibody to corresponding positions in a human antibody are well known and that skilled artisan can determine the equivalent locations in a human antibody.

[0087] In some embodiments, the inhibitor is conjugated to E104 of an IgG light chain. In some embodiments, the inhibitor is conjugated to E105 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to D150 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to D151 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to E153 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to E154 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to D169 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to D170 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to D183 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to D184 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to E184 of an IgG light chain. In some embodiments, the inhibitor is conjugated to D / E185 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to D185 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to E185 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to E186 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to E187 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to E194 of an IgG light chain. In some embodiments, the inhibitor IS conjugated to E195 of an IgG light chain. In some embodiments, the light chain is kappa light chain. In some embodiments, light chain is lambda light chain. In some embodiments, the numbering is according to the EU index. In some embodiments, the numbering is as defined by the EU index. In some embodiments, theEU index is as defined by Kabat. In some embodiments, the numbering is with respect to IgG2. In some embodiments, the numbering is with respect to a mouse antibody. The conversion of positions in a mouse antibody to corresponding positions in a human antibody are well known and that skilled artisan can determine the equivalent locations in a human antibody.

[0088] By another aspect, there is provided a composition comprising the ADC of the invention.

[0089] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a therapeutic composition. In some embodiments, the composition is for use in treating a disease. In some embodiments, the composition is for use in treating fibrosis. In some embodiments, the composition is for use in reducing fibrosis fibrosis. In some embodiments, the composition is for use in treating a disease comprising fibrosis. In some embodiments, the composition is for use in treating a disease characterized by fibrosis. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient or adjuvant.

[0090] As used herein, the term “carrier,” “excipient,” or “adjuvant” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxicpharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelies, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0091] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0092] In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is in need of treatment. In some embodiments, the subject suffers from a disease. In some embodiments, the subject suffers from fibrosis. In some embodiments, the composition is formulated for systemic administration. In some embodiments, the composition is formulated for local administration.

[0093] As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include parenteral, subcutaneous, oral, intramuscular, intratumoral, intraocular and intraperitoneal.

[0094] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0095] By another aspect, there is provided a method of reducing extracellular oxidase activity in a subject, the method comprising administering to the subject an ADC of the invention or a composition of the invention, thereby reducing extracellular oxidase activity.

[0096] By another aspect, there is provided a method of reducing extracellular oxidase activity in a composition comprising cells, the method comprising contacting the composition with an ADC of the invention or a composition of the invention, thereby reducing extracellular oxidase activity.

[0097] By another aspect, there is provided a method of reducing myofiber necrosis in a subject, the method comprising administering to the subject an ADC of the invention or a composition of the invention, thereby reducing myofiber necrosis.

[0098] By another aspect, there is provided a method of stabilizing myofibers in a subject, the method comprising administering to the subject an ADC of the invention or a composition of the invention, thereby stabilizing myofibers.

[0099] By another aspect, there is provided a method of treating fibrosis in a subject in need thereof, the method comprising administering to the subject an ADC of the invention or a composition of the invention, thereby treating fibrosis.

[0100] By another aspect, there is provided a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an ADC of the invention or a composition of the invention, thereby treating a disease.

[0101] In some embodiments, oxidase activity is lysyl oxidase activity. In some embodiments, oxidase activity is amine oxidase activity. In some embodiments, lysyl oxidase activity is the activity of a member of the lysyl oxidase gene family. In some embodiments, lysyl oxidase activity is LOX activity. In some embodiments, lysyl oxidase activity is selected from: LOX activity, LOXL1 activity, LOXL2 activity, LOXL3 activity, LOXL4 activity and a combination thereof. In some embodiments, lysyl oxidase activity is LOXL1 activity. In some embodiments, lysyl oxidase activity is LOXL2 activity. In some embodiments, lysyl oxidase activity is LOXL3 activity. In some embodiments, lysyl oxidase activity is LOXL4 activity.

[0102] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition or method herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life. In some embodiments, treating comprises reducing fibrosis. In some embodiments, treating comprises decreasing myofiber necrosis. In some embodiments, treating comprises stabilizing myofibers. In some embodiments, treating comprises increasing cardiac function. In some embodiments, cardiac function is measured by fractional shortening. In some embodiments, cardiac function is measured by ejection faction. In some embodiments, treating comprises increasing skeletal muscle function. In some embodiments, skeletal muscle function is measured by walking / running (e.g., treadmill test). In some embodiments, the skeletal muscle is the gastrocnemius.

[0103] In some embodiments, the fibrosis is selected from pulmonary fibrosis, liver fibrosis, renal fibrosis, peritoneal fibrosis, muscle fibrosis, skin fibrosis, pancreatic fibrosis, vascular fibrosis, cardiac fibrosis and systemic fibrosis. In some embodiments, the fibrosis is pulmonary fibrosis. In some embodiments, the fibrosis is liver fibrosis. In some embodiments, the fibrosis is renal fibrosis. In some embodiments, the fibrosis is peritoneal fibrosis. In some embodiments, the fibrosis is muscle fibrosis. In some embodiments, the muscle is skeletal muscle. In some embodiments, the skeletal muscle is the gastrocnemius. In some embodiments, the skeletal muscle is the diaphragm. In some embodiments, themuscle is smooth muscle. In some embodiments, the muscle is cardiac muscle. In some embodiments, the fibrosis is skin fibrosis. In some embodiments, the fibrosis is pancreatic fibrosis. In some embodiments, the fibrosis is vascular fibrosis. In some embodiments, the fibrosis is cardiac fibrosis. In some embodiments, the fibrosis is systemic fibrosis. In some embodiments, systemic fibrosis is cystic fibrosis. In some embodiments, the fibrosis is cystic fibrosis. In some embodiments, the fibrosis is selected from muscle fibrosis and cardiac fibrosis. In some embodiments, the fibrosis is selected from skeletal muscle fibrosis and cardiac fibrosis.

[0104] In some embodiments, treating fibrosis comprises treating the disease. In some embodiments, the disease is characterized by fibrosis. In some embodiments, the disease comprises fibrosis. In some embodiments, the disease is a fibrotic disease. In some embodiments, at least one symptom of the disease is fibrosis. In some embodiments, the disease is selected from the group consisting of: muscular dystrophy, cystic fibrosis, systemic sclerosis, scleroderma, thyroid-associated orbitopathy, cancer, inflammatory bowel disease, and Crohn’s disease. In some embodiments, the disease is muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne’s muscular dystrophy (DMD). In some embodiments, the muscular dystrophy is Becker muscular dystrophy. In some embodiments, the disease is cystic fibrosis. In some embodiments, the disease is systemic sclerosis. In some embodiments, the disease is scleroderma. In some embodiments, the disease is thyroid-associated orbitopathy. In some embodiments, the disease is cancer. In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the disease is inflammatory bowel disease (IBD). In some embodiments, the IBD is Crohn’s disease. In some embodiments, the disease is Crohn’s disease. The above recited diseases are all known to feature fibrosis which is mediated by extracellular lysyl oxidase activity. As such, all of these diseases can be treated by the ADCs and compositions of the invention. In some embodiments, the disease is in an early stage. In some embodiments, the disease is in a late stage.

[0105] In some embodiments, reducing comprises a reduction of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 87, 99 or 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, reducing comprises a reduction of at least 50%. In some embodiments, reducing comprises a reduction of at least 80%. In some embodiments, reducing is significantly reducing. In some embodiments, reducing is by at least a predetermined threshold.

[0106] By another aspect, there is provided a method of producing an antibody drug conjugate (ADC), the method comprising: a. providing an inhibitor of lysyl oxidase activity; b. providing an antibody or antigen binding fragment thereof; and c. conjugating the inhibitor to the antibody or antigen binding fragment thereof; thereby producing an ADC.

[0107] By another aspect, there is provided a method of producing an antibody drug conjugate (ADC), the method comprising: a. screening agents for their ability to inhibit lysyl oxidase activity and selecting at least one agent that is an inhibitor of lysyl oxidase activity; b. selecting a surface marker of M2 macrophages and producin an antibody or antigen binding fragment thereof that binds to the selected surface marker, or screening antibodies or antigen binding fragments thereof for their ability to bind the selected surface marker and selecting an antibody or antigen binding fragment thereof that binds; and c. conjugating the at least one agent to the produced or selected antibody or antigen binding fragment thereof; thereby producing an ADC.

[0108] In some embodiments, the agent is a compound. In some embodiments, the compound is an inhibitor. In some embodiments, the inhibitor is a small molecule inhibitor. In some embodiments, the inhibitor is an inhibitor of LOX, LOXL1, LOXL2, LOXL3 and / or LOXL4. In some embodiments, the inhibitor is an inhibitor of at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4. In some embodiments, the inhibitor is an inhibitor of LOX. In some embodiments, the inhibitor is not a protein inhibitor. In some embodiments, the inhibitor comprises a primary amide. In some embodiments, inhibiting lysyl oxidase activity is inhibiting LOX, LOXL1, LOXL2, LOXL3 and / or LOXL4. In some embodiments, inhibiting lysyl oxidase activity is inhibiting at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4. In some embodiments, inhibiting lysyl oxidase activity is inhibiting LOX.

[0109] In some embodiments, the inhibitor is selected from the group consisting of: CCT365623, PXS-5153A, Beta-aminopropionitrile (BAPN), aminomethylenepyridine 2(AMT), 2-aminomethylene-5-sulfonyl-AMT, 2-aminomethylene-4-sulfonyl-AMT, Trifluoromethyl (CF3)-substituted aminomethylene-pyridine, taurine, benzylamine, isoniazid, semicarbazide and thiosemicarbazide. In some embodiments, the inhibitor is CCT365623. In some embodiments, a library of agents is screened. In some embodiments, a library of compounds is screened. In some embodiments, a library of inhibitors is screened.

[0110] In some embodiments, the method further comprises testing inhibition of lysyl oxidase by the inhibitor. In some embodiments, the method further comprises testing inhibition of LOX by the inhibitor. In some embodiments, the method further comprises testing reduction in fibrosis by the inhibitor. In some embodiments, the method comprises providing an inhibitor, testing lysyl oxidase activity in the presence of the inhibitor and selecting an inhibitor that reduces lysyl oxidase activity. In some embodiments, the inhibitor is a plurality of inhibitors.

[0111] In some embodiments, the antibody binds a protein in fibrotic regions. In some embodiments, the antibody binds a protein specific to fibrotic regions. In some embodiments, the antibody binds a protein more highly expressed in fibrotic regions than non-fibrotic regions. In some embodiments, the antibody binds a protein of M2 macrophages. In some embodiments, the antibody binds a surface protein of M2 macrophages. In some embodiments, the antibody binds the selected surface protein of M2 macrophages. In some embodiments, the protein is a marker for M2 macrophages. In some embodiments, binds is specifically binds. In some embodiments, binds is specific to. In some embodiments, binds to a protein is against the protein. In some embodiments, the antigen of the antibody is the protein. In some embodiments, the protein is the target protein. In some embodiments, the method comprises selecting a surface marker of M2 macrophages.

[0112] In some embodiments, the method comprises producing an antibody or antigen binding fragment thereof that binds to the selected surface protein. In some embodiments, the method comprises producing an antibody or antigen binding fragment thereof specific to the surface protein. Methods of producing antibodies are well known in the art and include immunizing animals with the protein / marker or a fragment thereof (i.e., the extracellular domain of the protein / marker). In some embodiments, obtaining the antibody or antigen binding fragment thereof comprises immunizing an organism with the extracellular domain or fragment thereof of the selected M2 macrophage marker, and collecting antibodies from the immunized organism. In some embodiments, the organism is a mouse. In some embodiments, the organism is selected from a rabbit, a mouse, a rat, a shark, a camelid, a chicken a goat and a phage. In some embodiments, the camelid is selected from a camel anda llama. In some embodiments, the collecting comprises drawing blood. In some embodiments, the collecting comprises: a. extracting B cells from a spleen of the immunized organism; b. fusing the extracted B cells with myeloma cells to produce a hybridoma; and c. collecting antibodies from the hybridoma.

[0113] In some embodiments, screening is screening a library of antibodies or antigen binding fragments thereof. In some embodiments, a library is a list. Lists of antibodies are available online and may be screened for antibodies that binds the selected marker / protein. In some embodiments, the library is a phage display library. In some embodiments, the library is an immunized library derived from splenic B cells. In some embodiments, the library is an IgG library. In some embodiments, the IgG is IgG2. In some embodiments, the IgG2 is IgG2a.

[0114] In some embodiments, the antibody is an anti-CD206 antibody. In some embodiments, the antibody is an anti-CD163 antibody. In some embodiments, the antibody is antibody 2A6A10. In some embodiments, the antibody is antibody with the same CDR as 2A6A10, the same heavy chain and light chain variable regions as 2A6A10 or which competes with 2A6A10 for binding to CD206. In some embodiments, the method further comprises testing binding of the antibody to the target protein. In some embodiments, the method further comprises testing binding of the antibody to M2 macrophages. In some embodiments, the method further comprises testing binding of the antibody to fibrotic tissue. In some embodiments, the method comprises providing an antibody, testing binding to the protein and selecting an antibody that binds to the protein. In some embodiments, the antibody is a plurality of antibodies.

[0115] In some embodiments, the conjugating is linking. In some embodiments, the conjugation is covalent conjugation. In some embodiments, the conjugation is with a linker. In some embodiments, the conjugation is without a linker other than a single bond. In some embodiments, the conjugation is by an amide bond. In some embodiments, the conjugating comprises EDC / NHS coupling. In some embodiments, the conjugating is via an EDC / NHS coupling reaction. In some embodiments, the EDC / NHS coupling comprises linking an acid to a primary amide. In some embodiments, a primary amide is a primary amine. In some embodiments, the primary amide is part of the inhibitor. In some embodiments, the inhibitor comprises a primary amide. In some embodiments, the acid is an acidic amino acid. In someembodiments, an acidic amino acid is an acidic amino acid side chain. In some embodiments, the acid is the carboxylic acid end of an antibody chain. In some embodiments, the acid is the carboxylic acid end of an antibody chain. In some embodiments, the acid is an aspartic acid (D) or a glutamic acid (E) residue. In some embodiments, the acid is part of the antibody. In some embodiments, the acidic amino acid is an amino acid of the antibody. In some embodiments, the acid is an E or D base of the antibody.

[0116] In some embodiments, the conjugating comprises conjugating a plurality of inhibitors to the antibody or antigen binding fragment thereof. In some embodiments, the conjugating comprises conjugating a plurality of the inhibitor to the antibody or antigen binding fragment thereof. In some embodiments, the conjugating comprises conjugating a plurality of inhibitor molecules to the antibody or antigen binding fragment thereof. In some embodiments, multiple copies of the same inhibitor are conjugated to the antibody or antigen binding fragment thereof. In some embodiments, multiple copies are a plurality of inhibitors. In some embodiments, at least two different inhibitors are conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the different inhibitors inhibit different lysyl oxidases.

[0117] In some embodiments, a plurality of inhibitor molecule comprises between 2-100, 2- 90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-25, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13,2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40,3-30, 3-25, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3- 6, 3-5, 3-4, 4-100, 4-90, 4-80, 4-70, 4-60, 4-50, 4-40, 4-30, 4-25, 4-20, 4-19, 4-18, 4-17, 4- 16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-100, 5-90, 5-80, 5-70, 5-60,5-50, 5-40, 5-30, 5-25, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9,5-8, 5-7, 5-6, 6-100, 6-90, 6-80, 6-70, 6-60, 6-50, 6-40, 6-30, 6-25, 6-20, 6-19, 6-18, 6-17,6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-100, 7-90, 7-80, 7-70, 7-60, 7-50,7-40, 7-30, 7-25, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, or 7- 8 molecules of the inhibitor. Each possibility represents a separate embodiment of the invention. In some embodiments, the between 3-10 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof. In some embodiments, between 5-6 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof. In some embodiments, the between 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-25, 1- 20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1- 3, 1-2, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-25, 2-20, 2-19, 2-18, 2-17, 2-16, 2- 15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30, 3-25, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10,3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-100, 4-90, 4-80, 4-70, 4-60, 4-50, 4-40, 4-30, 4-25, 4-20, 4-19,4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-100, 5-90, 5- 80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-25, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12,5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-100, 6-90, 6-80, 6-70, 6-60, 6-50, 6-40, 6-30, 6-25, 6-20, 6- 19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-100, 7-90, 7-80, 7- 70, 7-60, 7-50, 7-40, 7-30, 7-25, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, or 7-8 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof. Each possibility represents a separate embodiment of the invention.

[0118] In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 95 or 100 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, about 5 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof. In some embodiments, about 6 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof.

[0119] In some embodiments, the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 5 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof. In some embodiments, at least 6 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof.

[0120] In some embodiments, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 100 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, at most 10 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof. In some embodiments, at most 6 molecules of the inhibitor are conjugated to the antibody or antigen binding fragment thereof.

[0121] In some embodiments, the method further comprises testing the conjugate. In some embodiments, the testing is after the conjugating. In some embodiments, the method comprises producing a plurality of ADCs and testing the plurality. In some embodiments,the method comprises selecting an ADC from the plurality of the ADCs. In some embodiments, the ADC is selected based on the testing.

[0122] In some embodiments, the testing is testing binding of the produced ADC to the target protein. In some embodiments, the testing is testing binding of the produced ADC to M2 macrophages. In some embodiments, the testing is testing binding of the produced ADC to fibrotic tissue. In some embodiments, the testing is testing targeting of the produced ADC to M2 macrophages. In some embodiments, the testing is testing targeting of the produced ADC to fibrotic tissue. In some embodiments, the method comprises selecting an ADC that binds. In some embodiments, the method comprises selecting an ADC that targets. In some embodiments, binds is specifically binds. In some embodiments, targets is specifically targets.

[0123] In some the testing is testing inhibition of lysyl oxidase activity. In some the testing is testing inhibition of LOX. In some the testing is testing inhibition of LOXL1. In some the testing is testing inhibition of LOXL2. In some the testing is testing inhibition of LOXL3. In some the testing is testing inhibition of LOXL4. In some the testing is testing inhibition of at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4. In some the testing is testing inhibition of LOX. In some embodiments, testing inhibition is testing extracellular inhibition. In some embodiments, the testing is testing in the extracellular milieu. In some embodiments, the testing is testing in fibrotic tissue. In some embodiments, fibrotic tissue is in an animal. In some embodiments, fibrotic tissue is in subject. In some embodiments, the method comprises selecting an ADC that inhibits.

[0124] In some embodiments, inhibits is specifically inhibits. In some embodiments, inhibits is significantly inhibits. In some embodiments, inhibits is inhibits by at least a predetermined threshold. In some embodiments, inhibits comprises a reduction of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 87, 99 or 100% of activity. Each possibility represents a separate embodiment of the invention. In some embodiments, inhibits is at least as strongly as the inhibitor inhibits when not conjugated to the antibody or antigen binding fragment thereof. In some embodiments, inhibits is comparable inhibition to the inhibitor inhibits when not conjugated to the antibody or antigen binding fragment thereof. In some embodiments, comparable comprises a variance of less than 35, 30, 25, 20, 15, 10, or 5%. Each possibility represents a separate embodiment of the invention. In some embodiments, comparable comprises a variance of less than 25%. In some embodiments, comparable comprises a variance of less than 10%.

[0125] In some embodiments, the testing is testing reducing fibrosis. In some embodiments, the testing is testing myofiber necrosis. In some embodiments, the testing is testing stabilization of myofibers. In some embodiments, the testing is in vitro. In some embodiments, the testing is in vivo. In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the testing is testing improving fibrosis. In some embodiments, the testing is testing treating fibrosis. In some embodiments, reducing fibrosis is inhibiting fibrosis.

[0126] In some embodiments, both binding and inhibition are tested. In some embodiments, an ADC that both binds and inhibits is selected. In some embodiments, the best binder is selected. In some embodiments, the best inhibitor is selected. In some embodiments, the best binder and inhibitor is selected. In some embodiments, the ADC that provides the best combination of binding and inhibition is selected.

[0127] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0128] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0129] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0130] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0131] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0132] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0133] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0134] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0135] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0136] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I- III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Materials and Methods

[0137] Mice: All experiments involving mice conform to the relevant regulatory standards. All mice are housed in IVC’s (Techniplast) according to space requirements defined by the NRC. All rooms are set to have 22 °C ± 2° and humidity of 30-70%. All HVAC parameters are controlled by a central computerized monitoring system. Light cycle is set to full light 10 h half-light 2 h and complete darkness 12 h, light cycle is monitored by the computerized central system.

[0138] Cell Culture: The RAW264.7 macrophage cell line was cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum, 1% pen-strep, 1% glutamine, 1% Pyruvate (Biological Industries) at 37°C and 5% CO2. To induce polarization into M2 macrophages, RAW264.7 cells were cultured in Serum- free media, 1% pen-strep, 1% glutamine, 1% Pyruvate supplemented with 20 ng / mL interleukin (IL)-4 and 20 ng / mL IL- 13 (PeproTech) for 48 hours. Fibroblasts were isolated from C57BL / 10 mdx mice and cultured in DMEM with 10% fetal bovine serum. Human Dermal fibroblasts (HDFc) (originated from male)were cultured in Fibroblast Growth Medium (FGM2 C-23020, PromoCell). Human Aortic Smooth Muscle Cells (HAOSMC) were cultured as previously described in Aviram, et al., 2024, “Coordination between cytoskeletal organization, cell contraction and extracellular matrix development, is depended on LOX for aneurysm prevention”, bioRxiv, 2024.02.23.581837, the contents of which is hereby incorporated by reference in its entirety.

[0139] Lentiviral Infections: HDFc infection, using viral particles generated in HEK293T cells using CalFectin Mammalian Cell Transfection Reagent (SignaGen, SL100478). MISSION® shRNA lentiviral plasmids (Merck) were used for knockdown of LOX (shLOX - TRCN0000045991) and a non-target shRNA was used for control (shCtrl - SHC016). Viral particles generated by the HEK293T cells were collected in Fibroblast Growth media for 48 hours and HDFc were infected. 24 hours post infection, the media was replaced and Puromycin selection was administered for the following 3 days.

[0140] Visualizing cell morphology: HDF knockdown of LOX Cells (shLOX) or non-target shRNA cells (shCtrl) were seeding at a density of 8xl0A4 per 1cm2. HDFc were seeding at a density of 8xl0A4 per 1cm2and incubated with Lox inhibitor (CCT365623, AOBIOUS, Cat#AOB37362), ADC, or control (DMSO, D2650 Merck) for 48 hours.

[0141] Flow Cytometry: RAW246.7 cells were freshly harvested and labeled with specific mAbs at 4°C in the dark for 30 minutes for flow cytometric analysis. Cells were first washed with FACS buffer containing PBS + 0.5% BSA. After brief centrifugation of the antibody stocks, the antibodies were diluted to the desired concentration in FACS buffer. The cells were then incubated with these fluorochrome-labeled antibodies for 30 min at 4°C. After staining, cells were washed, resuspended in FACS buffer, strained and acquired on a 5 Laser BD LSRFortessa cytometer running DIVA v9.4 (BD Biosciences) with standard manufacturer optical configuration. Single stained controls were acquired using Compbeads (BD Biosciences). The antibody that was used is CD206-AF647 (MR5D3; BD Biosciences).

[0142] Dot plot overlay was generated with FlowJo 10.10 (BD Biosciences). The total cell population was gated based on forward versus side scatter followed by doublet discrimination gates (FCS-w / FCS-H). The single cell population was analyzed for CD206 expression.

[0143] Immunofluorescence staining: Cells and cryosections of flash frozen gastrocnemius, diaphragm, and heart muscles slides were fixed with 4% paraformaldehyde solution (36% stock, Sigma-Aldrich, 50-00-0, diluted with PBS, 1:9) and permeabilized with 0.1% Triton X-100 in PBS (PBT 0.1%). Fixed cells or fresh frozen tissue cryosections were incubatedover-night at 4°C with primary antibodies. The following antibodies were used: anti-CD206 (Proteintech, Cat No: 60143- 1-Ig, 1:100); eMyo (DSHB, Cat No: F.1652, 1:200); Laminin 2 (Sigma Alderich, Cat No: L0663); Collagen I (Abeam, Cat No: ab21286, 1:200); IgG (Jackson ImmunoResearch Laboratories, Cat No: 159750, 1:200); anti-Pdgfra (R&D, Cat No: AF1062, 1:100); anti-CD31 (Dianova, Cat No: DIA-310, 1:100); anti-ki67 (Invitrogen, Cat No: 14-5698-82, 1:100); anti-F4 / 80 (Biolegend, Cat No: 123102, 1:100); and DAPI (Biolegend, 422801, 1:10,000) were also used. Secondary antibodies were incubated for 2 hours at room temperature. Secondary antibodies used: Alexa Fluor 594 mouse (Jackson ImmunoResearch Laboratories, Cat No: 715-585-151, 1:400); Alexa Fluor 488 mouse (Jackson ImmunoResearch Laboratories, Cat No: 715-545-151, 1:400); Cy3 rat (Jackson ImmunoResearch Laboratories, Cat No: 712-165-153, 1:400). Paraffin sections of nonmuscle tissues (lung, kidney and liver) were fixed with 4% paraformaldehyde solution (36% stock, Sigma-Aldrich, 50-00-0, diluted with PBS, 1:9) and permeabilized with 0.1% Triton X-100 in PBS (PBT 0.1%). Tissues were incubated over-night at 4°C with primary antibody anti-E-Cadherin (Sigma Alderich, Cat No: U3254, 1:200).

[0144] Masson Trichrome stain: Diaphragm, heart and gastrocnemius sections were first fixed in 4% paraformaldehyde (PFA) then washed three times with phosphate-buffered saline (PBS). The tissues were then subjected to a tissue processing procedure using a Tissue Processor machine (Leica TP1020, Germany). Next tissues were embedded in Paraplast paraffin to form a solid block. 4pm sections were produced using a microtome (Leica RM2265 Rotary Microtome, Germany). Subsequently, the sections were deparaffinized in xylene, rehydrated, and subjected to Masson’s Tri chrome Stain.

[0145] Immunohistochemistry : Immunohistochemistry was performed on fresh frozen slides using the Mouse and Rabbit Specific HRP / DAB IHC Detection Kit-Micro-polymer (Abeam, ab236466). Briefly slides were covered with hydrogen peroxide block and incubated at room temperature for 10 minutes. This was followed by a PBS wash to remove unbound hydrogen peroxide. Non-specific binding was blocked by incubating slides in protein block for 10 minutes at room temperature, then slides were washed 1 time in PBS. Slides were then incubated with a rabbit monoclonal anti-collagen I antibody (Abeam, Cat No: ab21286, 1:200 dilution) overnight at 4°C. This was followed by 3 washes in PBS to remove unbound primary antibody. After washing, slides were incubated with goat antirabbit HRP-conjugate for 15 minutes. Slides were then washed 4 times in PBS to remove unbound secondary antibody-HRP conjugate. Antibody binding was visualized using DAB substrate chromogen solution. Sections were counterstained with eosin aqueous solution 1%(Kaltek, Cat No: 1119) for 5 minutes followed by a 1-minute tap water wash to remove excess eosin. Slides were mounted using an aqueous non-fluorescing mounting media (National Diagnostics, Cat No: HS-106) and coverslips were applied. Collagen 1 staining was identified as brown staining and was evaluated under a light microscope.

[0146] Reagents: The LOX inhibitor CCT365623 was purchased from (AOBIOUS, Cat No: AOB37362), 3 -Aminopropionitrile fumarate salt (BAPN) (Sigma- Aldrich, Cat No: A3134), N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS) (FH24507; Tzamal D-Chem Laboratories Ltd, Israel), N-(3-dimethylaminopropyl)-N’-ethyl carbodiimide hydrochloride (EDC) (03450; Sigma- Aldrich), 12-14 kDa dialysis membrane (132 700; Repligen, US), and Human CD206 / CLEC13D PicoKine® Quick ELISA Kit (EK2049; BOSTER, US).

[0147] ADC Synthesis: The ADC was prepared using EDC / NHS coupling reaction. First, 1.01 mg mL-1 of anti-CD206 antibody (PBS), 2 mg / mL of Sulfo-NHS (PBS), and 2 mg / mL of EDC (DMSO) solutions were prepared. Second, each reagent solution was added to the antibody solution to reach a 1:25 molar proportion (antibody :Sulfo-NHS) and 1:10 molar proportion (antibody:EDC). Using 1.2 M HCL, the pH of the antibody solution with reagents was adjusted to 6. Third, the solution was mixed (450 rpm) at 25 °C for 1 h. Fourth, the antibody solution was dialyzed against PBS (pH 6; 1:1000 volume ratio) using a 12-14 kDa dialysis membrane (132700; Repligen) at 4 °C overnight to remove excess reagents. Fifth, ADC molecules were synthesized by mixing antibody activated solution with 2 mg / mL of LOX inhibitor (PBS) solution at a molar proportion of 1:10. Sixth, the pH of the reaction solution was adjusted to 8.4 with IM sodium bicarbonate, and the reaction was incubated for 2 hours at 450 rpm and maintained at 25 °C. Last, non-conjugated LOX inhibitor was removed by dialysis against PBS (pH 7.4; 1:1000 volume ratio) using a 12-14 kDa dialysis membrane at 4 °C; the external buffer was exchanged three times (after 1 hour, 4 hours, and overnight, respectively). The final ADC product was stored at -80°C until use.

[0148] In order to determine the quantity of LOX inhibitor linked to the anti-CD206 antibody in the ADC product, absorbance measurements were taken at 275 nm using a plate reader for the absorbance of the ADC, the anti-CD206 antibody alone, and a combination of the anti-CD206 antibody (non-activated) and 10 equivalents of the LOX inhibitor (initial inhibitor amount).The calculation of LOX inhibitor molecules per antibody molecules was carried out using the following equations: m (LOX inhibitor amount conjugated to anti-CD206 antibody) =n (LOX inhibitor) = m (LOX inhibitor amount conjugated to anti-CD206 antibody) / Mw (LOX inhibitor) = X [mole]Units LOX inhibitor = n (LOX inhibitor) [mole] x N [mole-1] = Z n (anti-CD206 antibody) = m (reaction amount of anti-CD206 antibody) / Mw (anti-CD206 antibody) = Q [mole]Units anti-CD206 antibody = n (anti-CD206 antibody) [mole] x N [mole-1] = WLOX inhibitor molecules per anti-CD206 antibody molecules in ADC product = Units (LOX inhibitor) / Units (anti-CD206 antibody) = 5-6

[0149] Mass spectrometry proteomics analysis: Native, non-conjugated CD206 (control) and ADC samples were subjected to a rigorous analysis employing Discoverer 2.4 identification with the Sequest (Thermo) search algorithm. The samples underwent trypsin digestion and were analyzed by LC-MS / MS using a Q-Exactive HF instrument (Thermo). Protein identification was performed with the Discoverer software against the mouse Uniprot database, with the incorporation of CCT365623 as a variable modification on the proteins' C-Terminus or specific amino acids (glutamic and aspartic). Peptides exclusive to the control sample were highlighted in red, while those exhibiting reduced abundance were categorized into orange, yellow, and green based on ADC / control mAb ratios. Identified proteins were filtered for high confidence, top rank, mass accuracy, and stringent criteria, including a 1% False Discovery Rate (FDR) threshold were applied. Semi-quantitation was achieved by calculating peak areas, providing a robust methodology for uncovering and understanding the differential protein conjugation patterns in ADC.

[0150] ELISA assay: Various ADC samples were synthesized by altering the molar equivalents of CCT365623 inhibitor (IN) molecules conjugated to native non-conjugated CD206 antibodies. The samples were stored at -80°C until activity ELISA measurements were conducted. Controls included native CD206 mAb and a solution of native CD206 mAb with 100 equivalents of IN that were not chemically linked. A 384-well plate was coated with 10 pg / mL of each sample, sealed, and incubated overnight at 4°C. The next day, the plate was washed three times with TBS-T wash buffer. Following this step, the Human CD206 / CLEC13D PicoKine® Quick ELISA Kit was used according to the manufacturer's protocol, excluding the coated strip microplate. For signaldevelopment, a color-developing reagent (TMB) was used, and kinetic absorbance was measured using a plate reader at 650 nm at 2-minute intervals for 1 hour. Absorbance values were analyzed at a chosen time point. The mean absorbance value for the CD206 mAbs (control) was calculated and normalized to 100%. The absorbance values of the other samples were then normalized to the control mean and expressed as percentages.

[0151] Nano Differential Scanning Fluorimetry (nanoDSF): In thermal unfolding experiments, 10 pL of 1.10 mg / mL (three replicates) of CD206 native and ADC samples were loaded into UV transparent capillaries and mounted in a Prometheus NT.48 (both from Nanotemper Technologies, Germany). The temperature gradient was set to rise l°C / min in the range 20-95 °C. Protein unfolding was measured by detecting the temperaturedependent change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm. Melting temperatures were determined by detecting the maximum of the first derivative of the fluorescence ratios (F350 / F330). Data was processed using the ThermControl software (Nanotemper Technologies, Munich, Germany).

[0152] Cell Proliferation Assay: Cell proliferation of M2 macrophages was quantified using the XTT Cell Proliferation Assay (Cat No: 4891-025-K). Following polarization, M2 macrophages were seeded at a density of 3xl0A4 cells / mL per well in 96-well microplates. Cells were incubated for 2 hours. Then, 50pl of XTT solution was added to each well, followed by an initial plate reading (T=0). Treatments (Untreated, ADC, anti-CD206, CCT365623, no cell control) were then added to the appropriate wells, with 6 wells used for each treatment condition. Absorbance readings were taken at 490 nm, with a reference wavelength of 630nm, at specific timepoints: 0, 1, 2, 4, 6, and 8 hours post-treatment. This time course allowed for the monitoring of M2 macrophage proliferation and treatment effects over an 8 -hour period. All treatments were normalized to the control containing only medium and XTT without cells.

[0153] RNA Isolation and cDNA Synthesis: Cell pellets were incubated with TRIzol reagent (Invitrogen, Cat No: 15596026) to obtain cell content. RNA was isolated using phase separation with Chloroform. RNA was then precipitated with isopropanol and washed with 75% EtOH. Following RNA isolation, the RNA was reverse transcribed and cDNA was prepared using cDNA kit (Abeam, Cat No: G592) and then proceeded to Eppendorf Mastercycler X50a (PCR system) for cDNA synthesis.

[0154] Real-time Quantitative PCR: qPCR was performed using SYBR green reagents on a StepOnePlus Real-Time PCR System (Applied Biosystems, Cat No: 4376600). The PCRpackage in R statistical software was used to calculate relative gene expression levels from qPCR data using the comparative AACt method, with GAPDH as the reference gene for normalization. Data is presented as bar graphs showing mean ± SD, with bar height representing normalized relative expression levels (fold change) and error bars denoting within-sample variability. The Syber green primer probes used are listed in Table 3 below.

[0155] Table 3: Primers used

[0156] In Vivo Studies: All experiments involving mice were performed according to the relevant regulatory standards (Technion IACUC and national animal welfare laws, guidelines, and policies). All mice are housed in IVC’s (Techniplast) according to space requirements defined by the NRC. All rooms are set to have 22 ± 2 °C and humidity of 30- 70%. HVAC parameters and light cycle are set by the computerized central system to full light of 10 hours, half-light of 2 hours and complete darkness of 12 hours. All mice were bred on a C57BL / 10ScSnJ background. For efficacy studies, male mdx mice were randomly assigned to groups (n<6 per group) receiving weekly intraperitoneal injections of either ADC (20ug / once a week / lOOul) or control treatment (20ug / once a week / lOOul) for 3 or 4 months (3-6 or 6-10 months respectively). Muscles were harvested at the experimental endpoint for further analyses.

[0157] Treadmill Running Test: Locomotion was assessed using a treadmill running test. The treadmill belt was set to 0° inclination. Electrical shock intensity of the resting pad was set to 0.3 mA. Mice were exercised on the treadmill twice per week for 40 minutes over the 3- or 4-month period. Each session involved a 10-minute warm-up period followed by a 30- minute running test. For warm-up, mice ran at 4 m / min for 2 minutes then 8 m / min for 8minutes. For the running test, mice ran at a constant speed of 12 m / min. The test was completed when the animal stopped on the resting pad. Distance travelled was calculated using the formula: Ax = (v) x (t); where Ax is the distance run, (v) is the constant velocity of 12 m / min, and (t) is the time elapsed before the mouse stopped. Locomotion was assessed by measuring the total distance travelled during each 30-minute running test session over the 3-4 month period.

[0158] Confocal microscopy: Confocal microscopy was performed at the Biomedical Core Facility with a Zeiss LSM88O system (Carl Zeiss SAS, Jena, Germany), equipped with a GaAsp PMT and 2 multi alkali PMTs. Images were acquired with a 20x0.8 objective, image pixel size was 120 nm. The following fluorescence settings were used: DAPI (excitation 405; emission 410-480), GFP (489; 490-535), Cy3 (561; 570-695). Sequential scanning was used for better channel separation. GFP and Cy3 were recorded with GaAsp detector, DAPI with multi alkali PMT.

[0159] Olympus Fluorescence Microscopy: Microscopy imaging was performed with an Olympus BX3-CBH system equipped with cellSens 1.18 software (OLYMPUS CORPORATION, Tokyo, Japan). Images were acquired with a 20x objective. The following fluorescence settings were used: DAPI (excitation 405; emission 410-480), GFP (excitation 488; emission 490-535), Cy3 (excitation 561; emission 570-620), and Cy5 (excitation 633; emission 640-740).

[0160] Olympus Brightfield Microscopy: Brightfield microscopy imaging was performed with an Olympus CKX41 microscope equipped with cellSens 1.18 software (OLYMPUS CORPORATION, Tokyo, Japan). Images were acquired with the built-in 6V30WHAL halogen light source and a lOx objective (PlanCN, N.A. 0.25). The LBD filter was used for neutral color balance.

[0161] Echocardiography - Vevo3100: Cardiac analysis was done by conventional two- dimensional imaging and M-Mode recordings using VevoLab software (FujiFilm, VisualSonics). Maximal left ventricular end-diastolic (LVDd) and end-systolic (LVDs) were measured in short-axis M-mode images. Fractional shortening (FS) and Ejection fraction (EF) were calculated.

[0162] Automatic slide scanner: Slides were digitalized using a Panoramic Flash 250 digital slide scanner (3DHistech, Budapest, Hungary) in bright-field mode using a x20 / 0.8 objective lens, pixel size 0.242nm with the extended focus mode to select the sharpest image from 3 focal planes spaced 1 pm apart.

[0163] Graphs and Statistical Analysis: All statistical analyses and graph plotting were performed using R statistical software. Normality of data distributions was assessed using the Shapiro-Wilk test. Homogeneity of variances was evaluated with Levene's test. For two- group comparisons of normally distributed data with equal variances, an unpaired two- sample t-test was performed. The non-parametric Mann- Whitney U test was used for nonnormal data or data failing tests of parametric assumptions. One-way ANOVA was applied to examine differences between three or more independent groups meeting normality and variance homogeneity criteria. Kruskal-Wallis tests were applied for non-parametric comparisons of multiple independent groups. Dunn's post-hoc tests with Bonferroni correction were used for pairwise comparisons where applicable. Two-sample Kolmogorov- Smirnov tests were used to compare histogram distributions. Data are expressed as mean ± SD. Data are presented as box plots indicating the median, interquartile ranges, and outliers beyond 1.5 times the interquartile range are shown as individual dots. Statistical significance was defined as p<0.05. Precise p-values are denoted as **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05 or non- significant (ns) based on the statistical test. Graphs represent mean + SEM.

[0164] Image Analysis and Quantification: All image processing and quantitative analysis was performed using Python version 3.10. Digital images of stained tissue sections and cell cultures acquired via fluorescent and confocal microscopy as well as Automatic slide scanner, were imported into the Python environment. Relevant modules from scientific computing libraries such as OpenCV, NumPy, SciPy, and scikit-image were utilized for image segmentation, object identification and feature extraction. Custom Python scripts employing object-oriented and modular programming principles were developed to conduct automated image analysis in a reproducible manner. Algorithms for contour tracing, morphological measurements, and statistical analyses were implemented such as Cellpose. Parameters including collagen deposition percentages, myofiber morphometries, cellular metrics and marker counts were extracted computationally.Example 1: Generation of anti-CD206 / LOX inhibitor ADC

[0165] LOX is a key ECM-modifying enzyme essential for the maintenance of multiple tissues. Its activity is also highly associated with numerous fibrotic diseases, including DMD. Since LOX has crucial intracellular activities required for various processes such as cellular proliferation and differentiation, it is critical to target its inhibition specifically to the extracellular matrix of fibrotic regions. One effective approach is using an antibody-drugconjugate (ADC), which is targeted to fibrotic regions within tissues while carrying a LOX inhibitor.

[0166] CD206 expressing M2 pro -regenerative macrophages populate fibrotic regions in multiple diseases and tissues, including fibrotic muscles in human DMD patients and mdx mice. Targeting these macrophages can deliver the drug to diseased regions. To test this, immuno staining analysis with an anti-CD206 antibody was carried out on muscles from healthy control mice (C57B110 / ScSn) and fibrotic muscles (cardiac, diaphragm, and gastrocnemius) from mdx mice. It was found that CD206 localized specifically to fibrotic regions highlighted with Collagen type I (Coll) immuno staining (Fig. 1A-1F) confirming that the anti-CD206 antibody targets the diseased regions. CD206 is an endocytic receptor. It was next tested whether, after antibody binding, CD206 remains on the cell surface without immediate endocytosis by macrophages. The murine macrophage cell line RAW264.7 was stimulated with interleukins IL-4 and IL- 13 to induce M2 polarization. Live M2 macrophages were incubated with the anti-CD206 antibody and fixed after 1, 4, or 8 hours. One hour after adding the antibody, minimal antibody endocytosis is observed, and M2 macrophage membranes are highly decorated with the antibody (Fig. 1G; yellow arrows). However, after 4 hours, most of the antibody was internalized and found intracellularly (Fig. 1G). These results suggest that the anti-CD206 antibody could serve as the targeting arm for an ADC to reach fibrotic tissues.

[0167] The ADC was generated by conjugating native CD206 mAb (2A6A10, Proteintech) to CCT365623 inhibitor (IN) molecules via stable covalent amide bonds using EDC / NHS coupling chemistry (Fig. 2A). The drug-antibody ratio of IN units to anti-CD206 units was calculated to be approximately 5-6, indicating that each anti-CD206 mAb was conjugated with 5-6 IN molecules (see methods). The sequence of the generic mouse IgG2A antibody (heavy chain SEQ ID NO: 21, light chain SEQ ID NO: 22, Fig. 2G) contains 58 glutamic acid and aspartic acid residues. The success of the conjugation process was evaluated using mass spectrometry proteomic analysis (Fig. 2B(i)). Five peptides were found only in the native CD206 sample and not in the ADC sample, indicating that these peptides were likely bound by the IN molecules and therefore not identified in the protein identification analysis. Specifically, these peptide fragments contain 11 binding sites (labeled in red) of amino acids (glutamic and / or aspartic, See Figure 2F) that were potentially conjugated by the IN molecules. When these linkages occur, the peptide cannot be recognized against the mouse Uniprot database. Other peptides categorized in orange, yellow, and green were found with varying frequencies according to the ADC / native CD206 mAbs ratio, with green indicatingthe highest similarity. The proposed 3D structure of the ADC is shown in Fig. 2B(ii), with potential binding sites to the IN molecules labeled in red. To study the bioactivity and binding affinity of the ADC, its binding with human MRC1 protein was examined using an ELISA assay (Fig. 2C). When the molar equivalents of IN molecules exceeded 10, the binding of the antibody was reduced by -50% (p < 0.0001) compared to the native CD206 mAb. With 10 equivalents of IN molecules, binding was reduced to 81.67+11.11% compared to the native CD206 (p = 0.0197). Significant destruction of the antibody's binding occurred only with 100 equivalents of IN. There was no significant difference in binding between the “100 equivalents of IN not chemically linked to CD206” sample and the “native CD206” sample. Consequently, the “10 equivalents of IN / CD206” ADC was utilized for subsequent experiments. To assess whether the conjugation process affected the structure and stability of the ADC compared to the native CD206, Nano Differential Scanning Fluorimetry (nanoDSF) measurements were performed (Fig. 2D). The minor thermal changes observed in the ADC indicate that the antibody remained intact after the conjugation, conjugation did not alter its structure, and the antibody stayed folded. Overall, these findings confirm that the conjugation of IN molecules to CD206 mAbs can be achieved with minimal impact on antibody structure and target binding, provided the ratio of IN is below a very high ratio (e.g., 100:1), thereby preserving the bioactivity and stability of the resulting ADC.

[0168] An ADC has two arms: it specifically targets the antigens it is raised against and maintains the conjugated drug activity. The observation that the conjugated LOX IN bound primarily to the Fc regions and not to the antibody’s antigen binding domains [Fab regions; Fig. 2B(ii)] suggested that the ADC could bind the CD206 receptor on M2 macrophages similarly to the unconjugated antibody, thus maintaining its specificity to M2 macrophages. To test this possibility, RAW264.7 cells were used and were polarized into M2 macrophages. FACS analysis demonstrates the polarization conditions induce -62% of the cells into becoming M2 macrophages (Fig. 2E). As expected, both the native anti-CD206 and the ADC did not bind the non-polarized macrophages (Fig. 3A). Notably, upon polarization into the M2 phenotype, both the anti-CD206 and the ADC showed specific binding to the cell membranes (Fig. 3B, yellow arrows).

[0169] Having confirmed that the ADC maintains its specificity to M2 macrophages, whether its other arm, i.e. LOX inhibition, was not compromised following conjugation was tested. Human dermal fibroblasts, which express high LOX levels and show significant reduction in cell size upon LOX knockdown (Fig. 3C) were used. Culturing the cells withfree CCT365623 or with the ADC, lead to a significant reduction in cell size in a like manner to the observed LOX knockdown (Fig. 3D), altogether suggesting that the ADC’s other arm, i.e., the activity of the conjugated molecule, is maintained.

[0170] Following the observation that the anti-CD206 is internalized already after 4 hours (Fig. IB), it was desired to test whether the ADC affects M2 macrophage viability or proliferation. Towards that end an XTT assay was carried out and these properties in untreated, ADC, -non-conjugated inhibitor- and non-conjugated anti-CD206-treated M2 macrophages were monitored. No differences in the viability or proliferation of the cells regardless of the treatments were observed (Fig. 3E). These results suggest that the ADC, even upon internalization, does not lead to M2 macrophage cell death.Example 2: ADC accumulates in fibrotic muscle lesions in mdx mice

[0171] Having demonstrated the specificity of the ADC in vitro, it was next tested whether it localizes to fibrotic tissues in vivo without affecting healthy tissues, similar to the free anti-CD206 (Fig. 1A). Since fibrotic diseases such as DMD affect multiple muscles, including the diaphragm, cardiac and skeletal limb muscles, treating such a disease requires a systemic approach. For such an anti-fibrotic drug, repeated administrations will be necessary. However, in mice, repeated tail vein injections are problematic, therefore the ADC was administered via intraperitoneal (IP) injections.

[0172] Six-month-old dystrophic mdx mice and age- and genetic background-matched control C57Bl / 10ScSn male mice were IP injected with 20pg ADC in lOOpl saline. Two hours later, the mice were harvested, and the tissues were sectioned and stained to identify the ADC using a secondary antibody against the anti-CD206 antibody. Fibrotic regions were identified using Coll immunostaining.

[0173] No ADC was observed in limb, diaphragm, or cardiac muscles in the C57Bl / 10ScSn control mouse (WT; Fig. 4A-C, right panels). In contrast, multiple foci of ADC staining were found in all dystrophic muscles of the mdx mouse (Fig. 4D-F, right panels, yellow arrowheads). To test if the ADC is specific only to the diseased tissues, the lungs, liver and kidneys of WT and mdx mice were further monitored for 24 hours following the injection. No ADC localization was found in these non-fibrotic regions, even in the diseased mdx mice (Fig. 4J), demonstrating that the ADC targets only the diseased fibrotic tissues.

[0174] The majority of CD206 expression has been reported in macrophages and dendritic cells, it has also been observed in liver endothelial cells but not in myogenic cells. Therefore,to further confirm the ADC's specificity, additional co-staining experiments were performed with markers for other cell populations. The above immuno staining analyses of the ADC itself or the anti-CD206 (Fig. 4A-F and Fig. 1A, respectively) demonstrated cells expressing the receptor localize to the interstitium. Therefore, ADC localization was compared to that of fibroblasts marked by PDGFRa, endothelial cells marked by CD31 / PECAM and F4 / 80, a pan-macrophage marker (Fig. 4G-I, middle panel). No ADC staining colocalized with that of fibroblasts or endothelial cells. In contrast, all foci marked by ADC were also positive for F4 / 80. Notably, ADC marked only a subset of the F4 / 80 expressing cells suggesting it targets some, but not all, types of macrophages.Example 3: ADC treatment improves muscle functionality in vivo

[0175] After confirming the ADC's in vivo targeting to M2 macrophages in fibrotic muscles following IP injection, next its effectiveness in inhibiting fibrosis and improving muscle functionality was assessed in dystrophic mdx mice. The aim was to determine whether ADC treatment could maintain or improve cardiac and skeletal muscle function and slow their deterioration over a period of 3 months. At the age of 3 months, mice were treated by weekly IP injections of either the ADC (20pg / 100pl; N=6), CCT365623 (16.3pg / 100pl N=5), unconjugated anti-CD206 (20pg / 100pl; N=5), IgG (a non-relevant antibody; 20pg / 100pl; N=5) or saline (N=4) and these were compared to WT (C57Bl / 10ScSn) non-treated mice (N=5). Twice a week, treadmill running tests (12 m / min / for 30 min) assessed skeletal muscle function, and cardiac function was monitored using echocardiography once a month. Mice were treated for 3 months and harvested at the age of 6 months.

[0176] Treadmill running tests show that already by the age of 3 months mdx mice experience reduced muscle function when compared to the WT mice (Fig. 5A). Treating the mdx mice with free CCT365623, unconjugated anti-CD206, IgG or saline had no effect, and the treadmill results of these mice resembled those of the untreated mdx mice (N=8). Strikingly, already following 3 weeks of ADC treatment, a significant improvement in the running capacity of the mdx mice was observed (Fig. 5A and Fig. 5D). Notably, although the ADC-treated mice did not attain the running capacity of the WT mice, no reduction in their treadmill running capacity was observed until the end of the experiment which was at the age of 6 months (Fig. 5A and 5D). These results suggest that muscle function was not only improved but was also maintained following ADC treatment.

[0177] Next, the impact of ADC treatment on cardiac function was assessed. M-mode images were used to calculate fractional shortening (FS) and ejection fraction (EF), measuresof cardiac contractility. Similar to the beneficial effects observed in the treadmill running assays, FS and EF of the ADC-treated mice were significantly improved compared to those of the control-treated (unconjugated free CCT365623, unconjugated anti-CD206, IgG or saline) or untreated mdx mice (Fig. 5B-5C, 5E-5F).

[0178] These results demonstrate that ADC treatment significantly improved muscle function, while free unconjugated anti-CD206 or CCT365623 had no effect, as mice treated with them behaved similarly to those treated with IgG, saline or non-treated ones. Altogether, these results suggest that the beneficial muscle phenotypes are not caused by the antibody’s simple targeting of the M2 macrophages or general LOX inhibition but rather due to the specific regionalized ADC activity.Example 4: ADC treatment reduces muscle fibrosis

[0179] The significant improvement of skeletal and cardiac muscle function following ADC treatment, along with the ADC’s targeting of fibrotic tissues, led to testing whether these beneficial effects could also be observed at the histological levels, specifically in reducing fibrosis. In mdx mice, the diaphragm is the most highly fibrosed muscle with early signs of fibrosis beginning at 1 month of age. By six months, the diaphragms of mdx mice are highly fibrosed. Cardiac fibrosis starts slightly later, at 3 months, while limb muscle fibrosis starts only at 6 months, i.e. at the end of this experiment. Therefore, a focus was placed on analyzing the fibrotic status of the diaphragm and heart by monitoring Coll immunostaining. Since all treatments apart from ADC behaved similarly, the ADC -treated mice were compared to those treated with IgG. Coll immunostaining showed a significant reduction of its expression in both diaphragms and hearts of ADC-treated mice (Fig. 6A-6F).

[0180] The reduction in Coll protein expression led to testing whether this fibrosis inhibition occurred at the protein level or earlier, at the RNA levels. The expression of TGFpi, a pro- fibrotic cytokine, Collal and fibronectin were monitored using real time RNA PCR (RT- PCR) in diaphragms (N=6) and cardiac (N=6) muscles. TGFpi expression was upregulated in ADC-treated mice in both muscle types. Collal expression was significantly upregulated in diaphragms but showed only a tendency towards upregulation in the heart. Fibronectin expression was not affected in either muscle by ADC treatment. Similarly, Lox expression and CD206 expression were not affected in ADC-treated mice (Fig. 6G). Altogether, these results suggest that the inhibition of the fibrotic reaction occurs at the protein level, consistent with ADC inhibition of LOX, a post-translational collagen modifying enzyme. These results further demonstrate that feedback mechanisms that promote collagen secretionare activated in response to the ADC. However, since this collagen is presumably not crosslinked, it is lost and does not remain in the tissue.Example 5: ADC entails a protective role in mdx mice

[0181] The observation that ADC-treated mice performed significantly better on the treadmill compared to non-treated or control-treated mice raised the hypothesis that ADC administration also benefits limb muscles, even though fibrosis is not yet prevalent in this tissue at this stage of the disease. Progressive myofiber necrosis is a central feature of DMD pathology and is observed in mdx muscles even before fibrosis appears. It was tested whether ADC treatment could affect myofiber viability in the limb muscles. Since the sarcolemma of necrotic myofibers is highly perforated, allowing the entry of multiple antibodies, immuno staining with only a secondary antibody can identify such fibers. It was found that while untreated or IgG-treated gastrocnemius muscles had a high percentage of necrotic fibers, hardly any such fibers were observed in ADC-treated mice (Fig. 7A-7D). The reduction in myofiber necrosis was also accompanied by reduced myofiber regeneration observed by the significant decrease in embryonic myosin heavy chain (MHC3) staining (Fig. 7E-7H).

[0182] The above results raised the hypothesis that the ADC stabilizes the myofibers, also independently of fibrosis. To test this possibility 3 months old mdx mice were treated as above for only three weeks, encompassing a full cycle of muscle regeneration. Following 3 weeks (i.e. 3 ADC injections), mice were harvested and the presence of necrotic myofibers was monitored. Strikingly and in contrast to the IgG-treated mice, no necrotic myofibers were observed in the ADC-treated ones (Fig. 7I-7J). Altogether these results reinforce the notion that ADC-treatment stabilizes the myofibers.

[0183] Repeated cycles of myofiber injury and regeneration result in a large fiber size distribution, which can be observed by monitoring their cross-section area (CSA). However, monitoring CSA in the diaphragm and gastrocnemius muscles showed no significant differences between the two treatments (Fig. 7K). Altogether, these results suggest that the ADC prevents muscle injury and associated necrosis even before fibrosis appears, helping stabilizing the muscle and the fibers within.Example 6: ADC activity in late disease stages

[0184] Given the beneficial effects of ADC treatment prior to or at the early stages of fibrosis onset, ADC effectiveness at later disease stages was tested next. Mice were treated weeklywith ADC (N=7) or IgG as a control (N=7). Treadmill training was conducted twice a week, and monthly echocardiography assessed cardiac function. Unlike the previous experiment conducted at 3-6 months, this experiment began at 6 months of age, with mice harvested at 10 months.

[0185] Treadmill training showed no significant differences between the IgG- and ADC- treated mice; both groups performed significantly worse than WT mice (N=6) (Fig. 8A). However, cardiac activity monitoring revealed that both FS and EF were better in the hearts of ADC-treated mice compared to IgG-treated ones (Fig. 8B-8C).

[0186] Next, muscle fibrosis was monitored using Masson’s trichrome staining (MTC). No differences in cardiac fibrosis were observed between ADC- and IgG-treated mice (Fig. 8F- 8G), but a trend of decreased fibrosis was noted in the diaphragm (p-value 0.02; p-adjusted value 0.07, Fig. 8D-8E). In the gastrocnemius muscle, ADC-treated mice had significantly less fibrosis than IgG-treated mice, with levels resembling those of WT mice (Fig. 8H-8I). Overall, these results suggest that ADC treatment improves the disease status even at late stages of the disease.

[0187] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. An antibody drug conjugate comprising a Lysyl Oxidase (LOX) inhibitor, Lysyl Oxidase homolog 1 (L0XL1) inhibitor, L0XL2 inhibitor, L0XL3 inhibitor or L0XL4 inhibitor conjugated to an antibody or antigen binding fragment thereof that binds specifically to a surface marker of M2 macrophages.

2. The antibody drug conjugate of claim 1, wherein said inhibitor is a LOX inhibitor.

3. The antibody drug conjugate of claim 1 or 2, wherein said inhibitor comprises a free primary amide and wherein said inhibitor is conjugated by forming an amide bond between said free primary amide and an acidic amino acid residue of said antibody.

4. The antibody drug conjugate of any one of claims 1 to 3, wherein said inhibitor comprises a molecular weight of at most 1000 Daltons.

5. The antibody drug conjugate of any one of claims 1 to 4, wherein said inhibitor is selected from the group consisting of: CCT365623, PXS-5153A, Betaaminopropionitrile (BAPN), aminomethylenepyridine 2 (AMT), 2-aminomethylene- 5-sulfonyl-AMT, 2-aminomethylene-4-sulfonyl-AMT, Trifluoromethyl (CF3)- substituted aminomethylene -pyridine, taurine, benzylamine, isoniazid, semicarbazide and thiosemicarbazide.

6. The antibody drug conjugate of claim 5, wherein said inhibitor is CCT365623.

7. The antibody drug conjugate of any one of claims 1 to 6, comprising between 3-100 molecules of said inhibitor conjugated to said antibody or antigen binding fragment thereof.

8. The antibody drug conjugate of claim 7, comprising 5-6 molecules of said inhibitor conjugated to said antibody or antigen binding fragment thereof.

9. The antibody drug conjugate of any one of claims 1 to 8, wherein said marker of M2 macrophages is selected from CD206 and CD 163.

10. The antibody drug conjugate of claim 9, wherein said marker of M2 macrophages is CD206.

11. The antibody drug conjugate of claim 10, wherein said antibody is antibody 2A6A10.

12. The antibody drug conjugate of claim 10, wherein said antibody comprises the same CDRs as antibody 2A6A10, comprises the same heavy chain variable region and the same light chain variable region as antibody 2A6A10 or competes with antibody 2A6A10 for binding to CD206.

13. The antibody drug conjugate of any one of claims 1 to 11, wherein said antibody is an IgG2a antibody.

14. The antibody drug conjugate of any one of claims 1 to 13, comprising at least one molecule of CCT365623 conjugated via an amide bond to an acidic amino acid residue of said antibody.

15. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1 to 14 and a pharmaceutically acceptable carrier, excipient or adjuvant.

16. The pharmaceutical composition of claim 15, formulated for systemic administration.

17. A method of reducing extracellular LOX activity in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition of claim 15 or 16, thereby reducing extracellular LOX activity.

18. A method of treating fibrosis in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition of claim 15 or 16, thereby treating fibrosis in a subject.

19. The method of claim 18, wherein said fibrosis is selected from pulmonary fibrosis, liver fibrosis, renal fibrosis, peritoneal fibrosis, skeletal muscle fibrosis, skin fibrosis, pancreatic fibrosis, vascular fibrosis, cardiac fibrosis and systemic fibrosis.

20. The method of claim 19, wherein said fibrosis is selected from skeletal muscle fibrosis and cardiac fibrosis.

21. The method of any one of claims 18 to 20, wherein said treating fibrosis is treating a disease selected from the group consisting of: muscular dystrophy, cystic fibrosis, systemic sclerosis, scleroderma, thyroid-associated orbitopathy, gastrointestinal cancer, inflammatory bowel disease, and Crohn’s disease.

22. The method of claim 21, wherein said disease is muscular dystrophy.

23. The method of claim 22, wherein said muscular dystrophy is Duchenne’s muscular dystrophy (DMD).

24. A method of producing an antibody drug conjugate (ADC), the method comprising: a. screening agents for their ability to inhibit at least one of LOX, L0XL1, L0XL2, L0XL3 and L0XL4 and selecting at least one agent that is an inhibitor of at least one of LOX, L0XL1, L0XL2, L0XL3 and L0XL4; b. selecting a surface marker of M2 macrophages and producing an antibody or antigen binding fragment thereof that binds to said selected surface marker or screening antibodies or antigen binding fragments thereof for their ability to bind to said selected surface marker and selecting an antibody or antigen binding fragment thereof that binds; and c. conjugating said selected at least one agent to said produced or selected antibody or antigen binding fragment thereof; thereby producing an ADC.

25. The method of claim 24, wherein step (a) comprises screening agents for their ability to inhibit LOX and selecting at least one agent that is an inhibitor of LOX.

26. The method of claim 24 or 25, wherein said inhibitor comprises a free primary amide and wherein said conjugating comprises forming an amide bond between said free primary amide and an acidic amino acid residue of said antibody.

27. The method of claim 26, wherein said conjugating comprises an EDC / NHS coupling reaction.

28. The method of any one of claims 24 to 27, comprising conjugating between 3-100 molecules of said inhibitor to said antibody.

29. The method of claim 28, comprising conjugating 5-6 molecules of said inhibitor to said antibody.

30. The method of any one of claims 24 to 29, further comprising testing binding of said produced ADC to M2 macrophages and selecting an ADC that binds to M2 macrophages.

31. The method of any one of claims 24 to 30, further comprising testing inhibition of at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4 by said ADC and selecting an ADC that inhibits at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4.

32. The method of claim 31, comprising selecting an ADC that inhibits at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4 at least as strongly as said inhibitor not conjugated to said antibody or antigen binding fragment thereof inhibits said at least one of LOX, LOXL1, LOXL2, LOXL3 and LOXL4.

33. The method of any one of claims 24 to 32, wherein said marker of M2 macrophages is selected from CD206 and CD 163.

34. The method of any one of claims 24 to 33, wherein said inhibitor is selected from the group consisting of: CCT365623, PXS-5153A, Beta- aminopropionitrile (BAPN), aminomethylenepyridine 2 (AMT), 2-aminomethylene-5-sulfonyl-AMT, 2- aminomethylene-4-sulfonyl-AMT, Trifluoromethyl (CF3)-substituted aminomethylene-pyridine, taurine, benzylamine, isoniazid, semicarbazide and thiosemicarbazide.

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