Compositions and methods for fibrosis

Abstract

An isolated polypeptide comprising an antigen-binding domain that specifically binds to the C-terminal telopeptide of the α2(I) chain of human collagen I is provided herein. In some embodiments, the polypeptide further comprises a collagen binding peptide and / or an antigen-binding domain comprising human antibody framework regions. Also provided are polynucleotides encoding the polypeptides and methods for treating diseases or disorders associated with excessive collagen fibril formation.

Description

[0001] Attorney Docket No: 205961-7120W01(00553)

[0002] COMPOSITIONS AND METHODS FOR FIBROSIS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S.

[0005] Provisional Patent Application No. 63 / 749,952 filed on January 27, 2025, which is herein incorporated by reference in its entirety.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with government support under AR082791 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0009] The Sequence Listing submitted herewith as a xml file named "205961-7120xx.xml," created on January 21, 2026 and having a size of 88,061 bytes, is herein incorporated by reference in its entirety.

[0010] BACKGROUND

[0011] Collagen I, the most abundant structural protein of connective tissues such as skin, bone, and tendon, is first synthesized as a precursor molecule, procollagen. The formation of collagen fibrils is initiated by enzymatic processing of procollagen to expose telopeptides, which engage in site-specific intermolecular interactions to drive collage self-assembly. In vivo, collagen fibrils are stabilized by the covalent cross-links formed between fibril-incorporated collagen molecules. Self-assembly of collagen molecules results in collagen fibrils, the main component of fibrotic lesions, particularly scarring.

[0012] Collagen / collagen binding that aggregates collagen molecules into the fibrils is mediated through the interaction of the C-terminal a 1(1) and oc2(I) telopeptides of one collagen molecule and the Triple-helical Telopeptide-Binding Region (T-TBR) of another binding partner. The T-TBR is located within an a 1(1) chain in the region flanked by resides 776 and 796 (Prockop, et al. (1998) J Biol Chem. 273, 15598-15604).Attorney Docket No: 205961-7120W01(00553)

[0013] Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. Types of fibrosis include, for example, pulmonary fibrosis (lungs), idiopathic pulmonary fibrosis (where the cause is unknown), cirrhosis (liver), endomyocardial fibrosis (heart), mediastinal fibrosis (soft tissue of the mediastinum), myelofibrosis (bone marrow), retroperitoneal fibrosis (soft tissue of the retroperitoneum), progressive massive fibrosis (lungs), nephrogenic systemic fibrosis (skin), Crohn's Disease intestine, keloid (skin), myocardial infarction (heart), scleroderma / systemic sclerosis (skin, lungs), arthrofibrosis (knee, shoulder, other joints) and some forms of adhesive capsulitis (shoulder).

[0014] Although the fibrotic changes seen in excessive scarring may be triggered in many ways, such as trauma, accidental injury, or surgical procedures, most of them are developed through fundamentally similar pathways that, in the end, lead to altering a number of functions of involved tissues. For instance, after surgery in the abdomen, the formation of excessive scar tissue around abdominal organs often interferes with their functionality. After plastic face surgery, the formation of excessive scar tissue frequently compromises the benefits of the surgery. Excessive scar formation also presents a major complication in the eye after glaucoma surgery performed to maintain a lamellar channel from the subconjunctival space to the anterior chamber. However, the excessive scar formation frequently closes this pressure-reducing channel, thereby forcing the intraocular pressure to rise (Addicks, et al. ( 9 3)' Arch Ophthalmol.

[0015] 101, 795-798). Yet another significant problem with excessive formation of fibrous deposits is the foreign body response to medical devices and materials implanted in the human body (Anderson, et al. (2008) Semin Immunol. 20, 86-100). Moreover, excessive scarring of the vocal folds may severely alter their ability to vibrate, thereby causing several voice disorders (Lim, et al. (2006) Ann Otol Rhinol Laryngol. 115, 921-929). Another medical problem of localized fibrosis is the formation of keloids, excessive scars for which no successful treatment methods exist. This scarring is an ongoing and rising problem; as keloids are more common in Americans of African and Asian descent, it is expected that in the near future the number of keloid cases in the USA will increase due to the foreseen rise in the percentage of these ethnic groups (Taylor, et al. (2002) J Am Acad Dermatol. 46, S41-62).Attorney Docket No: 205961-7120W01(00553)

[0016] There is a need for effective therapeutics and therapeutic methods to treat or prevent the excessive deposition of collagen fibrils that is characteristic of fibrotic processes, and / or to reduce localized and / or systemic fibrotic lesions.

[0017] SUMMARY

[0018] In one aspect, the present invention provides an isolated polypeptide comprising an antigen-binding domain that specifically binds to the C-terminal telopeptide of the ot2(I) chain of human collagen I, wherein the antigen-binding domain comprises: (a) a heavy chain variable region that comprises three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYTFTDYPLH (SEQ ID NO: 1); HCDR2 comprises the amino acid sequence WISTETGEPTYADD (SEQ ID NO: 2) or WIATETGEPTYADD (SEQ ID NO: 3); and HCDR3 comprises the amino acid sequence GYYYY (SEQ ID NO: 4); and (b) a light chain variable region that comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence KSSQSLLNSRTRKNNL (SEQ ID NO: 5); LCDR2 comprises the amino acid sequence WASTRES (SEQ ID NO: 6); and LCDR3 comprises the amino acid sequence KQSYNLWT (SEQ ID NO: 7).

[0019] In an embodiment, the polypeptide comprises (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 8 or SEQ ID NO: 9; and / or (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 10 or SEQ ID NO: 11.

[0020] In another embodiment, the polypeptide comprises (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the heavy chain variable region set forth in any one of SEQ ID NOs: 84-89; and / or (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 10 or SEQ ID NO: 11.Attorney Docket No: 205961-7120W01(00553)

[0021] In an embodiment, the polypeptide is a Fab, a single-chain variable fragment (scFv), or an antibody.

[0022] In an embodiment, the polypeptide comprises a human antibody framework region. In an embodiment, the polypeptide comprises a human antibody constant region. In some embodiments, the constant region is an IgG constant region. In some embodiments, the constant region is an IgG4 constant region.

[0023] In an embodiment, the polypeptide comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or 9.

[0024] In an embodiment, the polypeptide comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10 or 11.

[0025] In an embodiment, the polypeptide comprises an IgG4 heavy chain constant region and an IgG4 light chain constant region. In some embodiments, the IgG heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IgG light chain constant region comprises the amino acid sequence of SEQ ID NO: 13.

[0026] In an embodiment, the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or 15.

[0027] In an embodiment, the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 16 or 17.

[0028] In an embodiment, the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 16.

[0029] In some embodiments, the polypeptide comprises a collagen binding peptide (CBP) comprising the amino acid sequence of any one of SEQ ID NOs: 18-47. In some embodiments, the CBP comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CBP further comprises a linker on one or both ends of the CBP. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 48-59. In some embodiments, the polypeptide comprises a CBP-linker comprising the amino acid sequence of any one of SEQ ID NOs: 60-62. In some embodiments, the polypeptide comprises 2-5 CBPs. In some embodiments, the 2-5 CBPs comprise one or more linkers comprising the amino acid sequence of any one of SEQ ID NOs: 4.Attorney Docket No: 205961-7120W01(00553)

[0030] In an embodiment, the polypeptide comprises an IgG4 heavy chain constant region and an IgG4 light chain constant region. In some embodiments, the IgG heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 63 and the IgG light chain constant region comprises the amino acid sequence of SEQ ID NO: 66.

[0031] In some embodiments, the IgG heavy chain constant region and / or IgG light chain constant regions comprises a CBP-linker of any one of SEQ ID NOs: 60-62. In one embodiment, the CBP-containing IgG heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 63 or 64. In some embodiments, the CBP-containing IgG light chain constant region comprises the amino acid sequence of SEQ ID NO: 65.

[0032] In an embodiment, a polypeptide comprising the CBP comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NO: 66-69. In some embodiments, the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 70 or 71. In certain embodiments, the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or 67 and a light chain comprising the amino acid sequence of SEQ ID NO: 70.

[0033] In an embodiment, the polypeptide comprises an scFv comprising the heavy chain variable region and the light chain variable region. In some embodiments, the scFv comprises a linker between the heavy chain variable region and the light chain variable region. In some embodiments, the scFv comprises a linker connecting the carboxy terminus of the heavy chain variable region to the amino terminus of the light chain variable region. In other embodiments, the scFv comprises a linker connecting the carboxy terminus of the light chain variable region to the amino terminus of the heavy chain variable region. In certain embodiments, the scFv comprises a linker comprising the amino acid sequence of any one of SEQ ID NOs: 48-59. In some embodiments, the scFv comprises a linker between the heavy chain variable region and the light chain variable region, wherein the linker comprises the amino acid sequence of SEQ ID NO: 50, 58, or 59.

[0034] In another aspect, the present invention provides one or more polynucleotides or expression constructs individually or collectively encoding an a2(I)Ct binding polypeptide as described herein. In some embodiments, each of the expression constructs is derived from an expression vector. In some embodiments, the expression constructs encode a signal peptide. InAttorney Docket No: 205961-7120W01(00553)

[0035] some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 72-74.

[0036] In some embodiments, the expression constructs comprise a first expression construct encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 75 or 76, and a second expression construct encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO: 77 or 78. In one embodiment, the first expression construct encodes a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 76 and the second expression construct encodes a light chain variable region comprising the amino acid sequence of SEQ ID NO: 78.

[0037] In another aspect, the present invention provides a method of treating a disease or disorder associated with excessive collagen fibril formation in a subject, comprising administering to the subject an effective amount of an a2(I)Ct binding polypeptide as described herein. In some embodiments, the disease or disorder comprises scar formation. In some embodiments, the disease or disorder comprises fibrosis. In some embodiments, the fibrosis comprises pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid formation, myocardial infarction, scleroderma / systemic sclerosis, arthrofibrosis, or adhesive capsulitis. In some embodiments, the fibrosis results from a surgical procedure. In some embodiments, the surgery is abdominal surgery, plastic surgery, glaucoma surgery, or surgery for implantation of a medical implant or device.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0040] FIG. 1 : Schematic illustrating anti-a2(I)Ct blocking antibody (ACA)-based inhibition of collagen fibrillogenesis. A procollagen I molecule with procollagen N and C propeptides (Np and Cp), and N, and C telopeptides (Nt and Ct) is shown. The telopeptide binding region (TBR) is also indicated. The Ct-TBR binding drives collagen fibril formation, and blocking this binding interaction, including with the ACA, alters the fibrillogenesis process. Removal of propeptidesAttorney Docket No: 205961-7120W01(00553)

[0041] (here with LysC) triggers fibrillogenesis. Assembly of collagen molecules into a fibril is followed by crosslinking (\) of collagen molecules by lysyl oxidase (LOX). Mature fibrils, blocking the Ct-TBR binding with the ACA, and reduction of the number of fibrils and degradation of collagen molecules not incorporated into fibrils are also depicted.

[0042] FIGs. 2A-2B: SDS-PAGE analysis of humanized anti-a2(I)Ct antibodies comprising combinations of six variant heavy chains (H1-H6) and two variant light chains (L1-L2). (A) anti-a2(I)Ct antibodies comprising H1-H6 heavy chains in combination with an LI light chain under non-reducing (“NR”) or reducing (“R”) conditions. LB601 / 603 refers to the parental mouse anti-anti-a2(I)Ct antibody. (B) anti-a2(I)Ct antibodies comprising H1-H6 heavy chains in combination with an L2 light chain under non-reducing (“NR”) or reducing (“R”) conditions.

[0043] FIGs. 3A-3B: Procollagen I (Pro-I) and collagen I (Col-I) chains separated in a polyacrylamide gel. (A) Schematic views of intact procollagen I; collagen I that includes telopeptides; and collagen I in which the telopeptides were digested by pepsin. Symbols: Np, N-terminal propeptide; Nt, N-terminal telopeptide; Cp, C-terminal propeptide; Ct C-terminal telopeptide. Individual procollagen chains are also indicated. An asterisk (*) indicates the C-terminal telopeptide of the pro-a2(I) chain. This telopeptide includes the epitope for the antibody variants. (B) Coomassie blue-stained procollagen I (Pro-I) and collagen I (Col-I) chains separated in a polyacrylamide gel. The faint bands visible in the Pro-I lane represent products of degradation of procollagen chains; protein bands visible in the Col-I lane below the 50 kDa marker represent pepsin.

[0044] FIGs. 4A-4B: Interaction of anti-a2(I)Ct antibodies with (A) procollagen I chains or (B) collagen chains. (A) Western blot analysis of the interaction of the antibody variants with procollagen I chains. Based on the migration patterns of the procollagen chains (see FIG. 3D) it is apparent that all variants specifically interact with the pro-a2(I) and corresponding products of degradation. (B) Western blot analysis of the interaction of the antibody variants with collagen chains lacking telopeptide regions (procollagen I chains treated with pepsin to remove the telopeptide regions).

[0045] FIGs. 5A-5B: a2(I)-telopeptide binding to anti-a2(I)Ct antibodies as a function of antibody concentration. (A) Binding of anti-a2(I)Ct antibodies comprising H1-H6 heavy chains in combination with LI light chain (B) Binding of anti-a2(I)Ct antibodies comprising H1-H6 heavy chains in combination with L2 light chain.Attorney Docket No: 205961-7120W01(00553)

[0046] FIG. 6: Representative Octet-biosensor-based binding curves for determining binding kinetics of antibodies to a2(I)Ct, as shown in Table 3.

[0047] FIGs. 7A-7D: Humanized anti-a2(I)Ct antibodies block fibril formation in vitro.

[0048] Representative images at 4x magnification (A, B) and lOx magnification (C, D) of collagen fibrils formed in vitro in the presence of control human IgG (hlgG) (A, C) or the humanized anti-a2(I)Ct antibodies, H8L2 or H9L2. Bar = 100 pm.

[0049] FIG. 8: SDS-PAGE analysis of the pellet (P) and supernate (S) fractions showing collagen fibril (P) or collagen monomer (S) fractions. The fibrils were formed in vitro in the presence of the inhibitory antibodies (H8L2, H9L2) or non-inhibitory control hlgG, CTR (z.e., no antibodies)).

[0050] FIGs. 9A-9B: Analysis of collagen fibril (P) or collagen monomer (S) fractions formed in vitro in the presence of different concentrations of the inhibitory antibodies. (A) SDS-PAGE analysis of the pellet (P) and supernate (S) fractions showing collagen fibril (P) or collagen monomer (S) fractions formed in the presence of different concentrations of the inhibitory antibodies (H8L2, H9L2) or non-inhibitory control antibodies (hlg). (B) Amount of collagen fibrils (P) and monomers (S) expressed as a function of the P / S ratios.

[0051] FIGs. 10A-10C: Schematic of SwiMR expression system for generating producer cells for the humanized anti-a2(I)Ct antibodies. (A) Bicistronic expression of membrane-anchored reporter (MAR), e.g., GFP, was mediated by IRES downstream of the gene of interest (GDI), e.g., anti-a2(I)Ct heavy chain The IRES-MAR cassette was flanked by 2 LoxP sites for recognition of DNA recombinase Cre. After treating the cells with Cre, the sequence between LoxP sites is removed from the chromosome. (B) LB602-H9 expression construct for directing expression of an anti-a2(I)Ct heavy chain (H9) comprising an Fc region of human IgG4 (S228P / F234A / L235A) under the control of a human EFla promoter. The IRES-GFP cassette was place downstream of the heavy chain and was flanked by two LoxP sites for excision from the chromosome after screening. (C) LB604-VL2 (or “LB604-L2”) expression construct for directing expression of an anti-a2(I)Ct light chain (L2) under the control of a human EFla promoter. The top ~1% of the cells transfected with LB602-H9 or LB604-L2 having the highest GFP signal were isolated by FACS.

[0052] FIGs. 11A-1 IB: FACS sorting of high producing cells. (A) Stable pools of 44.42.2 CHO-S cells selected after transfection with LB602-H8 / LB604-L2. 1.43% of the cells in the windowAttorney Docket No: 205961-7120W01(00553)

[0053] P9 were sorted as shown. (B) Stable pools of 44.42.3 CHO-S cells selected after transfection with LB602-H9 / LB604-L2. 1.43% of the cells in the window P9 were sorted as shown.

[0054] FIGs. 12A-12B: Retention of the anti a2(I)Ct (AC A) antibodies containing collagen-binding peptide (CBP) within collagen-rich matrices formed in cell culture conditions. (A) Visualization of the collagen-rich matrix formed around cultured fibroblasts. The presence of collagen within fibrillar matrices was analyzed using collagen-specific antibodies conjugated with a red fluorophore. The collagen fibrils were observed using fluorescence microscopy. The presence of cells was analyzed by staining their nuclei with 4',6-diamidino-2-phenylindole (DAPI). (B) Western blot analysis of H-ACA (humanized anti-a2(I)Ct antibody with no CBP) and CBP-ACA variant (with CBP) extracted from collagen-rich cell layers presented in panel 12A. Symbols: Hy, LK, heavy and light ACA chains; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Mw, molecular weight (in kilo Daltons, kDa) markers.

[0055] FIG. 13 depicts an X-ray image of a rabbit operated on to induce arthrofibrosis. Symbols: T; tibia, F; femur, P; pump, Kw; Kirshner wire, Tc; tube connector, Rp; refill port.

[0056] FIG. 14 depicts a custom device to measure knee flexion contracture in rabbit knee joints. Symbols: Tb; tibial clamp, Fe; femoral clamp. Arrows indicate the rotation of the torque mechanism during the flexion (Flex) and extension (Ext) phases.

[0057] FIGs. 15A-15C depict microscopic images of a rabbit’s posterior knee capsule stained with H&E (A) and picrosirius red (B and C).

[0058] FIGs. 16A-16B: Electrophoretic separation of collagenous proteins, pepsin-extracted from the posterior knee capsule. (A) Collagen chains are separated in reducing conditions from the beginning of the electrophoretic run. In these conditions, monomeric collagen I and collagen III chains co-migrate. (B) Collagen proteins run in delayed-reduction conditions. In these conditions, monomeric collagen I and collagen III chains separate. Symbols: Mw, molecular mass markers; KDa, kilodaltons; Un and In, samples from uninjured (Un) and injured (In) PC capsules; DTT, dithiothreitol added at the beginning of electrophoresis; d-DTT, dithiothreitol added 15 minutes after starting electrophoretic run; , y, cross-linked complexes comprising collagen chains; al (III), alpha 1 chain of collagen III; al (I) and a2(I), alpha 1 and 2 chains of collagen I.

[0059] FIG. 17 shows CBC parameter measurement before the knee injury surgery and at various post-surgery time points. Symbols: WBC, white blood cells; NEU, neutrophils; LYM,Attorney Docket No: 205961-7120W01(00553)

[0060] lymphocytes; MONO, monocytes; EOS, eosinophils; BAS, basophils; NEU %, percentage of neutrophils; LYM %, percentage of lymphocytes; MONO %, percentage of monocytes; EOS %, percentage of eosinophils; BAS %, percentage of basophils; HCT, hematocrit; HGB, hemoglobin; RBC, red blood cells; MCV, mean corpuscular volume; RDW %, red blood cell distribution width; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet count; MPV, mean platelet volume.

[0061] FIGs. 18A-18E: Histology of tissues and organs. (A) Histology of the aorta, heart, skeletal muscle, and Achilles tendon. Symbols: Turn, tunica media; Myc, myocardium. (B) Histology of liver, lung, spleen, and thymus. Symbols: Hl, hepatic lobule; Al, alveolus; Ln, lymphoid nodule; Cr-Tc, cortex thymic cells. (C) Histology of the stomach, esophagus, intestines, and kidney. Symbols: Mm, mucous membrane; Sm, submucosa; Lp, lamina propia; Sse-k, stratified squamous epithelium-keratinized; Ig, intestinal glands; Rc, renal cortex. (D) Histology of brain tissues and the sciatic nerve. Symbols: ScWm, subcortical white matter; Odg, oligodendroglia; Pc, Purkinje cells; Ep, epineurium; Fs, nerve fascicle. (E) Histology of ovaries and testis. Symbols: Grn, granulosa cells; Ti, theca interna; Oc, oocyte; St, Seminiferous tubule.

[0062] FIGs. 19A-19B: Graphical representations of joint stiffness measurements in two mACA-treated rabbits, (A) and (B). The graphs display torque profiles of uninjured (Un, black curves) and injured (In, red curves) knees during the extension (ascending) and flexion (descending) phases (see FIG. 14). Corresponding dotted curves indicate knee angles at various torque levels. The knee angles of the uninjured and corresponding injured limbs at the maximum torque of 0.2 Nm were used to calculate flexion contracture (see FIG. 20).

[0063] FIG. 20: Flexion contracture in the mACA and mACA groups vs. CTR. The flexion contracture data were calculated as the ratio of maximal knee extension angles measured at 0.2 Nm torque (see FIGs. 14 and 19). The interquartile range, which spans from the 25th to the 75th percentile, determines the height of each box. The horizontal lines represent the means, while the whiskers delineate the SD values.

[0064] FIG. 21 shows a histological quantification of various populations of picrosirius-stained collagen fibrils formed within uninjured (Un) and injured (In) posterior capsules (PCs) from mACA-treated, ACA-treated, and control rabbits.

[0065] FIGs. 22A-22D: FTIR-based assays of PCs from the mACA-treated rabbits. (A) Unstained PC samples were observed in a light microscope attached to the FTIR system. TheAttorney Docket No: 205961-7120W01(00553)

[0066] arrows indicate the collagen-rich areas of PCs. Corresponding samples stained with H&E and picrosirius red corroborated the location of the collagen-rich regions in unstained counterparts. (B) A representative FTIR PC spectrum with fitted peaks (green line). The insert shows residuals describing the fit of the peaks. Also, the peaks used to assay the relative collagen amount (1064 cm'1v and 1338 cm'1v) and mature cross-links (1660 cm'1v and 1690 cm'1v) are indicated. (C, D) graphic representations of the FTIR-based results on the relative amount of collagen (C) and the maturity of collagen cross-links (D) in the PC scar tissue from mACA-treated, ACA-treated, and control (CTR) rabbits. The ratios were calculated based on the areas of the spectral peak corresponding to the sulphated GAGs (centered around 1064 cm1v) and the spectral peak corresponding to collagen (centered around 1338 cm1v). The maturity of collagen cross-links is expressed as the ratio of the area of the spectral peak corresponding to the mature trivalent PYR cross-link (centered around 1660 cm'1v) and the immature divalent deDHLNL cross-link (centered around 1690 cm'1v). The interquartile range, which spans from the 25th to the 75th percentile, determines each box. The horizontal lines represent the means, while the whiskers delineate the SD values.

[0067] FIGs. 23A-23B: Graphic representations of the FTIR-based results on the relative amount of collagen (A) and the maturity of collagen cross-links (B) in the PC scar tissue from mACA-treated, ACA-treated, and control (CTR) rabbits. The ratios were calculated based on the areas of the spectral peak corresponding to the sulphated GAGs (centered around 1064 cm1v) and the spectral peak corresponding to collagen (centered around 1338 cm1v). The maturity of collagen cross-links is expressed as the ratio of the area of the spectral peak corresponding to the mature trivalent PYR cross-link (centered around 1660 cm'1v) and the immature divalent deDHLNL cross-link (centered around 1690 cm'1v). The interquartile range, which spans from the 25th to the 75th percentile, determines each box. The horizontal lines represent the means, while the whiskers delineate the SD values.

[0068] FIGs. 24A-24B: Assays of collagen III: collagen I ratios in PCs isolated from the mACA-treated, ACA-treated, and control rabbits. (A) Collagen III: collagen I ratios in PCs from uninjured (Un) and injured (In) knee joints. (B) The In / Un ratios of the corresponding values presented in FIG. 24A. The interquartile range, which spans from the 25th to the 75th percentile, determines each box. The horizontal lines represent the means, while the whiskers delineate the SD values.Attorney Docket No: 205961-7120W01(00553)

[0069] DETAILED DESCRIPTION

[0070] Definitions

[0071] Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedence over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0072] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0073] That the disclosure may be more readily understood, select terms are defined below. The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0074] The term “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, “about” is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%.

[0075] The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chainsAttorney Docket No: 205961-7120W01(00553)

[0076] interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Unless otherwise specified herein, the terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies as well as derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.

[0077] The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to bind to an antigen e.g., a2(I)Ct) specifically. The antigenbinding function of an antibody can be performed by fragments of a full-length antibody.

[0078] Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include, for example: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL1 and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two F(ab)' fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and (v) a dAb fragment (Ward et al. (1989) Nature 241 :544-546), which consists of a VH domain. Furthermore, although the VL and VH domains are coded for by separate genes, they can be joined using a synthetic linker or spacer that enables them to be made as a single contiguous chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed (see e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448). A "CDR" or complementarity determining region is a region of hypervariability interspersed within regions that are more conserved, termedAttorney Docket No: 205961-7120W01(00553)

[0079] "framework regions" (FR). The CDRs comprise the antigen-binding determinants of an antibody. In embodiments of the anti- a2(I)Ct antibody, a2(I)Ct / CBP antibody, or antigen-binding fragment of the invention, the FRs may be identical to corresponding human germline sequences or may be naturally or artificially modified.

[0080] As used herein, the term "framework," when used in reference to an antibody variable region, refers to all amino acid residues outside the CDR regions within the variable region of an antibody. A variable region framework is generally a discontinuous amino acid sequence between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs. As used herein, the term "framework region" is intended to mean each domain of the framework (or framework segment) that is separated by the CDRs.

[0081] A “humanized antibody” refers to an antibody in which the complementarity-defining regions (CDRs) of an antibody of a non-human mammal, e.g., a mouse, are grafted to a human antibody. The variable domain of each antibody heavy chain and light chain comprises three CDRs; the intervening sequence segments are “framework segments.” Each variable domain is composed of four framework segments. In a humanized antibody, the framework segments are typically of human origin.

[0082] A “nucleic acid” or “polynucleotide” as utilized in the practice of the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. The nucleic acid or polynucleotide may comprise any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like. The nucleic acid or polynucleotide may comprise DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or doublestranded form, including homoduplex, heteroduplex, and hybrid states.

[0083] As used herein, the terms “single-chain variable fragment” and “scFv” are used interchangeably with reference to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH:VL heterodimer. In some embodiments, the heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding spacer or linker, which connects the C-terminus of the VH with the N-terminus of the VL, or the C-terminus of the VL with the N-terminus of the VH. The terms “spacer” and “linker” are used interchangeably herein. In some embodiments,Attorney Docket No: 205961-7120W01(00553)

[0084] the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VH - spacer - VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL - spacer - VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

[0085] As used herein, the term “subject” or “patient” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[0086] Unless otherwise indicated, the term "treating" as used herein means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, an indicated disease or disorder. The term "treatment" as used herein, unless otherwise indicated, refers to the act of treating. The term “treating" does not necessarily mean that the disease or disorder will, in fact, be eliminated.

[0087] The term "therapeutically effective amount" or "effective amount" means the amount of a subject compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that is being sought.

[0088] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0089] a2(I)Ct and a2(T)Ct / CBP Binding Polypeptides

[0090] Engineered antigen-binding polypeptides are provided that bind to the C-terminal telopeptide (Ct) of the a2-chain of collagen I (or “ot2(I)Cf ’). The engineered binding polypeptides inhibit the formation of collagen fibrils. Free collagen molecules that do not incorporate into fibrils are readily accessible for degradation by enzymes present in theAttorney Docket No: 205961-7120W01(00553)

[0091] extracellular space (Prockop and Fertala, J. Biol. Chem. 273 : 15598-15604 (1988); Chang et al., Diabetes 29:778-781 (1980)).

[0092] In one aspect, the present invention provides an a2(I)Ct binding polypeptide (e.g., anti-a2(I)Ct antibody or antigen-binding fragment thereof), which comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In an embodiment, the VH region comprises three heavy chain complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequences set forth in SEQ ID NO: 1;

[0093] HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2 or 3; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3. In an embodiment, the Vi. region comprises three light chain CDRs: LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequences set forth in SEQ ID NO: 4; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6. The a2(I)Ct binding polypeptide is capable of binding the C-terminal telopeptide of the oc2Ct chain of human collagen I.

[0094] In one embodiment, the a2(I)Ct binding polypeptide comprises: (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 8 or 9; and / or (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 10 or 11. In another embodiment, the a2(I)Ct binding polypeptide comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or 9 and / or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10 or 11.

[0095] In another embodiment, the polypeptide comprises (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the heavy chain variable region set forth in any one of SEQ ID NOs: 84-89; and / or (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 10 or SEQ ID NO: 11.Attorney Docket No: 205961-7120W01(00553)

[0096] In an embodiment, the a2(I)Ct binding polypeptide is a Fab, a single-chain variable fragment (scFv), or an antibody.

[0097] In an embodiment, the a2(I)Ct binding polypeptide comprises human antibody framework regions.

[0098] In an embodiment, the a2(I)Ct binding polypeptide comprises a human antibody constant region, such as an IgG constant region. In some embodiments, the human antibody constant region is an IgG4 constant region. In certain embodiments, the a2(I)Ct binding polypeptide comprises an IgG4 heavy chain constant region and an IgG4 light chain constant region. In some embodiments, the IgG4 heavy chain constant region comprises the amino acid substitutions, S228P / F234A / L235A. In one embodiment, the IgG heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 12 and the IgG light chain constant region comprises the amino acid sequence of SEQ ID NO: 13.

[0099] In another embodiment, the a2(I)Ct binding polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or 15 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 16 or 17. In another embodiment, the a2(I)Ct binding polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 16. In another embodiment, the a2(I)Ct binding polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 17. In another embodiment, the a2(I)Ct binding polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 16. In another embodiment, the a2(I)Ct binding polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0100] In another aspect, the a2(I)Ct binding polypeptide is a dual-targeted a2(I)Ct / CBP binding polypeptide (e.g., anti-a2(I)Ct / CBP antibody or anti-a2(I)Ct / CBP-binding fragment thereof) comprising an a2(I)Ct binding polypeptide as described above, which further comprises a collagen-binding peptide (CBP). In some embodiments, the CBP is fused to the C-terminus and / or N-terminus of an anti-u2(I)Ct antibody heavy chain. Alternatively, or in addition, the CBP may be fused to the C-terminus and / or N-terminus of an anti-a2(I)Ct antibody light chain. In some embodiments, the CBP is fused to an internal position in the anti-a2(I)Ct antibody heavyAttorney Docket No: 205961-7120W01(00553)

[0101] chain and / or light chain, such as before, after, or within a hinge region of the heavy or light chain region of the antibody. In some embodiments, the CBP comprises the amino acid sequence of any one of SEQ ID NOs: 18-47. In some embodiments, the CBP comprises the amino acid sequence of SEQ ID NO: 43.

[0102] In some embodiments, the CBP comprises a spacer (or linker) fused to one or both ends of the CBP. In some embodiments, the CBP comprises a spacer on one or both ends of the CBP. The spacer is typically rich in glycine for flexibility, as well as serine or threonine for solubility. The spacer can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of spacers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entirety. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) linkers or spacers such as (GS)n, GSG, (GSGGS)n (SEQ ID NO: 48), (GGGS)n (SEQ ID NO: 49), and (GGGGS)n (SEQ ID NO: 50), where n represents an integer of at least 1. Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 51), GGSGG (SEQ ID NO: 52), GSGSG (SEQ ID NO: 53), GSGGG (SEQ ID NO: 54), GGGSG (SEQ ID NO: 55), GSSSG (SEQ ID NO: 56), GGGGS (SEQ ID NO: 57), (G4S)2 (SEQ ID NO: 58), (G4S)3 (SEQ ID NO: 59), and the like. Those of skill in the art would be able to select the appropriate spacer sequence for use in the present invention

[0103] In some embodiments, the spacer comprises the amino acid sequence of any one of claims 48-59. In some embodiments, the a2(I)Ct / CBP binding polypeptide comprises a CBP-linker comprising the amino acid sequence of any one of SEQ ID NOs: 60-62. In some embodiments, the a2(I)Ct / CBP binding polypeptide comprises 2-5 CBPs. In some embodiments, the 2-5 CBPs comprise one or more spacers comprising the amino acid sequence of any one of SEQ ID NOs: 48-59 or a combination thereof.

[0104] In an embodiment, the a2(I)Ct / CBP binding polypeptide comprises a human antibody constant region, such as an IgG constant region. In some embodiments, the IgG constant region comprises one or more CBPs. In some embodiments, the a2(I)Ct / CBP binding polypeptide comprises a human IgG4 heavy chain constant region and a human IgG4 light chain constant region. In some embodiments, the IgG4 heavy chain constant region comprises the amino acid substitutions, S228P / F234A / L235A. In some embodiments, the IgG4 heavy chain constantAttorney Docket No: 205961-7120W01(00553)

[0105] region comprises a CBP, resulting in a fusion protein comprising the amino acid sequence of SEQ ID NO: 63 or 64. In some embodiments, the IgG4 light chain constant region comprises a CBP, resulting in a fusion protein comprising the amino acid sequence of SEQ ID NO: 65.

[0106] In some embodiments, the a2(I)Ct / CBP binding polypeptide comprises a CBP in an anti-a2(I)Ct / CBP heavy chain or an anti-a2(I)Ct / CBP light chain. In other embodiment, the a2(I)Ct / CBP binding polypeptide comprises a CBP in both the anti-a2(I)Ct / CBP heavy chain and the anti-a2(I)Ct / CBP light chain.

[0107] In certain embodiments, the u2(I)Ct / CBP binding polypeptide comprises an a2(I)Ct heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or 15, and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 70 or 71.

[0108] In one embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 70.

[0109] In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 71.

[0110] In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 70.

[0111] In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 71.

[0112] In other embodiments, the a2(I)Ct / CBP binding polypeptide comprises an a2(I)Ct / CBP heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 66-69, and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 16 or 17.

[0113] In one embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 16. In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 17.Attorney Docket No: 205961-7120W01(00553)

[0114] In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NO: 67 and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 16. In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NO: 67 and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0115] In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NO: 68 and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 16. In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NO: 68 and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0116] In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NO: 69 and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 16. In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NO: 69 and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0117] In some embodiments, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 66-69 and an anti-a2(I)Ct light chain comprising the amino acid sequence of SEQ ID NO: 70 or 71.

[0118] In one embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NOs: 66 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 70. In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NOs: 66 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 71.

[0119] In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NOs: 67 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 70. In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chainAttorney Docket No: 205961-7120W01(00553)

[0120] comprising the amino acid sequence of SEQ ID NOs: 67 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 71.

[0121] In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NOs: 68 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 70. In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NOs: 68 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 71.

[0122] In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NOs: 69 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 70. In another embodiment, the a2(I)Ct / CBP binding polypeptide comprises an anti-a2(I)Ct / CBP heavy chain comprising the amino acid sequence of SEQ ID NOs: 69 and an anti-a2(I)Ct / CBP light chain comprising the amino acid sequence of SEQ ID NO: 71.

[0123] In an embodiment, the a2(I)Ct binding polypeptide or a2(I)Ct / CBP binding polypeptide comprises a humanized antibody. In an embodiment, the humanized antibody is produced by “grafting” (or substituting) the six CDRs from the mouse-derived antibody into human antibody framework regions (the non-CDR regions in the VII or VL), utilizing a DNA fragment of a human antibody as a template. The human antibody template may be selected based on the composition of the humanized antibody variable region. For example, in certain embodiments, the framework region of a human antibody variable region having a high homology, e.g., greater than about 80%, or greater than about 90%, or greater than about 95%, with the framework regions of a parental (e.g., mouse) framework region is selected. The general genetic recombination procedure for producing humanized antibodies is described, for example, in Kettleborough et al, Protein Eng. 4(7):773-783 (1991), EP 125023, and WO 96 / 02576.

[0124] In an embodiment, a polynucleotide encoding the heavy chain and / or light chain of a desired antibody is designed in which nucleic acid(s) encoding the aforementioned CDRs are ligated within the framework regions encoding a human heavy chain or light chain antibody. The DNA sequence is synthesized by a polymerase chain reaction method using oligonucleotide primers which are designed to have regions overlapping the terminal regions of the CDRs and the framework regions. The resultant DNA is ligated to DNA encoding the human antibodyAttorney Docket No: 205961-7120W01(00553)

[0125] constant region, and the ligation product is integrated into an expression vector. The resulting expression construct is introduced into a host, thereby producing the humanized antibody. See, e.g., WO 96 / 02576.

[0126] Humanized antibodies may be prepared, as exemplified in Jones et al., Nature 321:522-525 (1986), which describes replacing the CDR regions in a human antibody with those from a mouse. Also see Riechmann, 1988, Nature 332:323-327; Queen et al, 1989, Proc. Nat. Acad. Sci. USA 86: 10029 (preparation of humanized antibody binding the interleukin 2 receptor); and Orlandi et al, 1989, Proc. Natl. Acad. Sci. USA 86:3833 (describing the cloning of immunoglobulin variable domains for expression by the polymerase chain reaction). The CDRs are grafted throughout the framework regions so that the CDRs can form a functional antigenbinding site. In some embodiments, one or more amino acids in the framework regions within the VH or VL are replaced with other amino acids so that the CDRs of the resulting humanized antibody can form an optimal antigen binding site providing e.g., increased expression and / or increased or decreased binding affinity.

[0127] In an embodiment, a humanized anti-a2(I)Ct antibody or a2(I)Ct / CBP antibody of the present invention comprises human antibody framework regions and may further comprise a human antibody constant region. In some embodiments, the human antibody constant region is a human IgG constant region. In some embodiments, the human antibody constant region is a human IgG4 constant region. In embodiments, the human antibody constant region can comprise any of the heavy or light chain constant regions of human antibodies, including, for example, yl, y2, y3 or y4 for heavy chains, and K for light chains.

[0128] In an embodiment, the a2(I)Ct or a2(I)Ct / CBP binding polypeptide comprises a singlechain variable fragment (scFv) comprising the VH region and the VL region. In some embodiments, the scFv comprises a spacer between the heavy chain variable region and the light chain variable region. As used herein, the terms “single-chain variable fragment” and “scFv” are used interchangeably with reference to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH:VL heterodimer. In some embodiments, the heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding spacer or linker connecting the C-terminus of the VH with the N-terminus of the VL or connecting the C-terminus of the VL with the N-terminus of the VH. The terms “spacer” and “linker” are used interchangeably herein. In someAttorney Docket No: 205961-7120W01(00553)

[0129] embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VH— spacer— VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL — spacer— VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

[0130] The spacer is typically rich in glycine for flexibility, as well as serine or threonine for solubility. The spacer can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of spacers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers such as (GS)n, GSG, (GSGGS)n (SEQ ID NO: 48), (GGGS)n (SEQ ID NO: 49), and (GGGGS)n (SEQ ID NO: 50), where n represents an integer of at least 1. Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 51), GGSGG (SEQ ID NO: 52), GSGSG (SEQ ID NO: 53), GSGGG (SEQ ID NO: 54), GGGSG (SEQ ID NO: 55), GSSSG (SEQ ID NO: 56), GGGGS (SEQ ID NO: 57), (G4S)2 (SEQ ID NO: 58), (G4S)3 (SEQ ID NO: 59), and the like. Those of skill in the art would be able to select the appropriate spacer sequence for use in the present invention.

[0131] Despite removal of the constant regions and the introduction of a spacer, scFv fragments retain the specificity of the original immunoglobulin. Single chain scFvs can be expressed from a nucleic acid comprising VH- and Vr-encoding sequences separated by a spacer, including but not limited to those comprising the amino acid sequence of any one of SEQ ID NOs: 48-59. In some embodiments, the scFv comprises a spacer between the VH and VL regions, wherein the spacer comprises the amino acid sequence of SEQ ID NO: 50, 58, or 59. In some embodiments, the spacer connects the carboxy terminus of the VH region to the amino terminus of the VL region. In other embodiments, the spacer connects the carboxy terminus of the VL region to the amino terminus of the VH region.

[0132] Amino acid sequences cited in this disclosure are described in Table 1 below.

[0133] Table 1: Amino Acid Sequences

[0134]

[0135] Attorney Docket No: 205961-7120W01(00553)

[0136]

[0137] Attorney Docket No: 205961-7120W01(00553)

[0138]

[0139] Attorney Docket No: 205961-7120W01(00553)

[0140]

[0141] Attorney Docket No: 205961-7120W01(00553)

[0142]

[0143] Attorney Docket No: 205961-7120W01(00553)

[0144]

[0145] Attorney Docket No: 205961-7120W01(00553)

[0146]

[0147] Nucleic Acids and Expression Constructs

[0148] In another aspect, the present invention provides one or more polynucleotides individually or collectively encoding an a2(I)Ct binding polypeptide or an a2Ct / CBP-binding polypeptide thereof as described herein.

[0149] Suitable vectors include, e.g., plasmids, expression vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example and should not be construed in any way as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR40, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL.

[0150] In some embodiments, one or more expression constructs are provided, which comprise any one of the polynucleotides described herein in one or more expression vectors. Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. In certainAttorney Docket No: 205961-7120W01(00553)

[0151] embodiments, a selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett etal., / «v^V. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0152] Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, an adeno-associated virus vector, an adenovirus vector, a poxvirus vector, a herpesvirus vector, a foamy virus vector, an engineered hybrid virus vector, a transposon-mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals.

[0153] In some embodiments, one or more expression constructs encode a signal peptide for secretion of the antibodies or antigen-binding fragments thereof. In some embodiments, a polynucleotide or expression construct of the present disclosure further comprises a leader sequence encoding a signal peptide at the N-terminal end of the encoded antibody, antigenbinding fragment, or fusion protein which is cleaved from the antigen binding domain during cellular processing and secretion of the polypeptide. Suitable signal peptide sequences are knownAttorney Docket No: 205961-7120W01(00553)

[0154] to those of skill in the art. In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 72-74.

[0155] In some embodiments, the expression vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e. ., a lentiviral vector) of the present invention.

[0156] In certain embodiments, the expression vector contains a signal peptide sequence, an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and / or one or more selectable markers.

[0157] Method for Producing Antibodies

[0158] In another aspect, the present invention provides a method for producing an a2(I)Ct or a2(I)Ct / CBP binding polypeptide (e.g., antibody) thereof as described herein.

[0159] Any suitable expression system may be used to produce the humanized antibody or antigen-binding fragment thereof, including those described above. Exemplary cells for expressing the humanized antibody or antigen-binding fragment include eukaryotic or animal cells, including e.g., established mammalian cell lines; fungal cells such as yeast cells; insect cells; and prokaryotic cells, including bacterial cells such as Escherichia coli. In certain embodiments, the humanized antibody or antigen-binding fragment is expressed in mammalian cells. The expression system may incorporate conventional promoters useful for the expression in mammalian cells, e.g., the human cytomegalovirus (HCMV) immediate early promoter.

[0160] Promoters for expression in mammalian cells may include virus promoters, such as those from retroviruses, lentiviruses, adenoviruses, polyoma viruses, SV40. Mammalian cell derived promoters include, for example, the human polypeptide chain elongation factor-1 alpha (HEF-1 alpha) promoter.

[0161] In one embodiment, two expression constructs are constructed, wherein the first expression construct comprises an a2(I)Ct VH region and an immunoglobulin constant region, and the second expression construct comprises an a2(I)Ct VL region and an immunoglobulin constant region.Attorney Docket No: 205961-7120W01(00553)

[0162] In certain embodiments, the first and / or second expression construct further encodes a collagen binding peptide (CBP) as described herein, such as a CBP comprising the amino acid sequence of any one of SEQ ID NOs: 18-47. In certain embodiments, the nucleic acid encoding the CBP is positioned within the heavy and / or light chain constant region(s), at the carboxy terminus of the heavy and / or light chain constant region(s), and / or at the carboxy terminus of the heavy and / or light chain constant region(s).

[0163] In some embodiments, the first expression construct encodes an a2(I)Ct VH regionhuman heavy chain constant region fusion protein under the control of expression control elements from a suitable enhancer / promoter system, and the second expression construct encodes an a2(I)Ct VL region-human light chain constant region fusion protein under the control of expression control elements from a suitable enhancer / promoter system. The human antibody constant region can comprise any of the heavy or light chain constant regions of human antibodies, including, for example, yl, y2, y3 or y4 for heavy chains, and K for light chains. In certain embodiments, the constant region comprises a human IgG4 heavy chain constant region comprising the amino acid sequence of SEQ ID NOs: 12, 63, or 64 and / or a human IgG4 light chain constant region comprising the amino acid sequence of SEQ ID NOs: 13 or 65.

[0164] In some embodiments, a single expression construct is used to express each of the anti-a2(I)Ct heavy and light chains from a single promoter-enhancer system. In one embodiment, a self-cleaving 2A peptide is fused in-frame between the heavy and light chains or an internal ribosome binding site (IRES) is positioned between the heavy and light chains for capindependent translation.

[0165] As used herein, a “self-cleaving 2A peptide” refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picomaviridae virus family, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.Attorney Docket No: 205961-7120W01(00553)

[0166] In some embodiments, the self-cleaving 2A peptide may comprise the T2A self-cleaving peptide of SEQ ID NO: 79, the P2A self-cleaving peptide of SEQ ID NO: 80, the E2A selfcleaving peptide of SEQ ID NO: 81, the F2A self-cleaving peptide of SEQ ID NO: 82, and / or multiple copies or combinations thereof. Those of skill in the art would be able to select the appropriate self-cleaving peptide for use in the present invention.

[0167] In some embodiments, the single polynucleotide or expression construct co-expressing the anti-a2(I)Ct heavy and light chains comprises an internal ribosome entry site (IRES) between the heavy and light chain coding regions. As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art, including, without limitation, IRES obtainable from viral or cellular mRNA sources, e. , immunoglobulin heavy-chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2 (FGF2); insulin-like growth factor (ILGF); translational initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). An exemplary IRES, encephalomyocarditis virus (EMCV) IRES, comprises the nucleotide sequence of SEQ ID NO: 83. Those of skill in the art would be able to select the appropriate IRES for use in the present invention.

[0168] In some embodiments, a method for producing an a2(I)Ct or a2(I)Ct / CBP binding polypeptide comprises construction of a single scFv expression construct coding for the anti-a2(I)Ct VH region and the anti-a2(I)Ct VL region, wherein each variable region is under the control of common expression control elements (e.g., enhancer / promoter system). In some embodiments, the scFv comprises a linker between the VH region and the VL region, such as a linker comprising the amino acid sequence of any one of SEQ ID NOs: 48-59. In some embodiments, the linker connects the carboxy terminus of the VH region to the amino terminus of the VL region. In some embodiments, the linker connects the carboxy terminus of the VL region to the amino terminus of the VH region. In some embodiments, the scFv further comprises a linker-CBP at the carboxy terminus of the scFv which comprises the amino acid sequence of SEQ ID NO: 61; a CBP-linker at the amino terminus of the scFv exemplified by the amino acidAttorney Docket No: 205961-7120W01(00553)

[0169] sequence of SEQ ID NO: 62; and / or a linker-CBP-linker positioned between the VH and VL, as exemplified by the amino acid sequence of SEQ ID NO: 60.

[0170] The one or two expression constructs above are used to transform a host cell. The transformed host is cultured in vivo or in vitro to produce a desired a2(I)Ct or a2(I)Ct / CBP binding polypeptide (e.g. , antibody or antigen binding fragment) secreted into the cell culture media. The a2(I)Ct or a2(I)Ct / CBP binding polypeptide may be isolated and purified from cell media according to well-known techniques. The concentration of the resulting antibody or antigen binding fragment can be determined by, for example, enzyme-linked immunosorbent assay (ELISA). Antigen binding activity can be confirmed by known methods, including ELISA, enzyme immunoassay, radioimmunoassay or fluorescent assay.

[0171] In certain embodiments, the present invention provides a producer cell comprising a stably transformed cell that expresses an antibody or antigen binding fragment described herein. As used herein, the term “producer cell” refers to a cell used for producing the antibody or antigen-binding fragment of the present invention. A producer cell can be a cell cultured in vitro, or a cell in vivo. Producer cells for generating the antibodies or antigen binding fragments of the present invention may include a variety of different primary cells and cell lines. In some embodiments, the producer cells are primary cells, such as primary mammalian cells, or cell lines derived therefrom. Exemplary cell lines for producing the antibodies or antigen binding fragments include NK92, HEK293, 293T, 293F, Chinese hamster ovary (CHO), CHO-S cells, and the like. In some embodiments, the producer cells are immune cells, or cell lines derived therefrom. Exemplary immune cells may include CAR-T cells, CAR-modified cytotoxic T cells, natural killer (NK) cells, dendritic cells, macrophages, lymphocytes (T cells or B cells), gamma delta T cells, alpha-beta T cells, mesenchymal stem cells (MSCs), bone marrow MSCs, umbilical cord MSCs, adipose-derived MSCs, dendritic cells, induced pluripotent stem cells (iPSCs), neutrophils, or any other cells of the immune system.

[0172] Methods of Use

[0173] In another aspect, the present invention provides a method of treating a disease or disorder associated with excessive collagen fibril formation in a subject, comprising administering to the subject an effective amount of an a2(I)Ct binding polypeptide or a2(I)Ct / CBP binding polypeptide as described herein. The a2(I)Ct binding polypeptide or a2(I)Ct / CBP binding polypeptide binds to the C-terminal telopeptides of the a2 chain of collagenAttorney Docket No: 205961-7120W01(00553)

[0174] I to inhibit the formation of collagen fibrils, and thereby prevent excessive deposition of collagen fibrils that is characteristic of fibrotic processes. The antibody or antibody fragments of the invention may thus be utilized to reduce localized and systemic fibrotic lesions, including but not limited to fibroses occurring in internal organs, the dermis, or the eye.

[0175] In some embodiments, the disease or disorder comprises scar formation. In some embodiments, the disease or disorder comprises fibrosis. In some embodiments, the fibrosis comprises pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid formation, myocardial infarction, scleroderma / systemic sclerosis, arthrofibrosis, or adhesive capsulitis. In some embodiments, the fibrosis results from a surgical procedure. In some embodiments, the surgery is abdominal surgery, plastic surgery, glaucoma surgery, or surgery for implantation of a medical implant or device.

[0176] The fibrotic lesion treated may be triggered by trauma, accidental injury or surgical procedures, among other causes. The antibody or antibody fragments of the invention may be administered, for example, after surgery in the abdomen to avoid the formation of excessive scar tissue around abdominal organs; after plastic surgery to the face to reduce scar formation; to the eye following glaucoma surgery performed to maintain a lamellar channel from the subconjunctival space to the anterior chamber, to prevent excessive scar formation that may function to closes the pressure-reducing channel and cause intraocular pressure to rise; and following implantation of medical devices and materials implanted in the human body, which would otherwise trigger a fibrotic response. In one embodiment, the antibody or antibody fragments are administered to treat or prevent the formation of keloids.

[0177] In some embodiments, the subject of treatment is human. In other embodiments, the subject is a veterinary subject. Treatment may involve administration of one or more antibodies or antigen binding fragments thereof as described herein, alone or with a pharmaceutically acceptable carrier. In some embodiments, the active agent(s) are administered in combination with one or more other therapeutic, diagnostic or prophylactic agents. In some embodiments, the active agent(s) are administered composition may thus further comprise an additional agent selected from the group consisting of corticosteroids, anti-inflammatories, immunosuppressants, antimetabolites, and immunomodulators, for example.Attorney Docket No: 205961-7120W01(00553)

[0178] The compositions of the invention may be administered parenterally. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0179] The compositions of the invention may be given orally, in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch.

[0180] Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried com starch.

[0181] The compositions of the invention may be administered topically. For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Topical-transdermal patches may also be used. For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.

[0182] Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0183] Topical or other localized administration is advantageously utilized at the site of a localized fibrosis, e.g., by localized injection. The location of the fibrosis may comprise, for example, a wound, particularly a wound edge.Attorney Docket No: 205961-7120W01(00553)

[0184] The dosage of active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. Detection and measurement of indicators of efficacy may be measured by a number of available diagnostic tools, including, for example, by physical examination including blood tests, pulmonary function tests, and chest X-rays; CT scan; bronchoscopy; bronchoalveolar lavage; lung biopsy and CT scan. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[0185] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0186] As a non-limiting example, an effective amount of an a2(I)Ct binding polypeptide or a2(I)Ct / CBP binding polypeptide is from about 0.025 to about 50 mg / kg, or from about 0.1 to about 50 mg / kg, or from about 0.1-25 mg / kg, or from about 0.1 to about 10 mg / kg, or from about 0.1 to about 3 mg / kg. Dosage may vary with the type and severity of the condition to be alleviated. For any particular treatment subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0187] Pharmaceutical

[0188]

[0189] and FormulationsAttorney Docket No: 205961-7120W01(00553)

[0190] Also provided are pharmaceutical compositions and formulations comprising the o.2(I)Ct binding polypeptide or a2(I)Ct / CBP binding polypeptide as described herein for treating subjects. Pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner with one or more pharmaceutically acceptable carriers or excipients. The antibodies or fragments thereof can be formulated for administration in accordance with the route of administration. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the antibody can be in lyophilized powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0191] The term "pharmaceutical composition" refers to a preparation that is formulated to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular EV and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10Attorney Docket No: 205961-7120W01(00553)

[0192] residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0193] The compositions used in the practice of the invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills, lyophilized cake, dry powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. In some embodiments, the a2(I)Ct binding polypeptide or a2(I)Ct / CBP binding polypeptide may be administered by using a pump, enema, suppository, or indwelling reservoir or such like.

[0194] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, lyophilized cake, dry powder, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile solutions can be prepared by incorporating the antibody or fragment in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization. In the case of sterile powders for the preparation of sterile solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile solution thereof. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. The desired characteristics of a solution can be maintained, for example, by the use of surfactants and the required particle size in the case of dispersion by the use of surfactants, phospholipids and polymers. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts, polymeric materials, oils and gelatin.

[0195] In certain embodiments, the a2(I)Ct binding polypeptide or a2(I)Ct / CBP binding polypeptide compositions of the invention may be prepared with a carrier that will protect the antibody against rapid release, such as a controlled release formulation, including implants,Attorney Docket No: 205961-7120W01(00553)

[0196] transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York (1978)).

[0197] In some embodiments, buffering agents are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0198] In some embodiments, the composition is formulated as an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the antibodies or antigen binding fragments, preferably those with activities complementary to the antibodies or antigen binding fragments, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition may further include other pharmaceutically active anti-fibrosis agents, including but not limited to pirfenidone, nintedanib, tocilizumab, hydronidone, ruxolitinib, mycophenolate mofetil / mycophenolic acid, decorin, a CD45 inhibitor, or a TGF-beta inhibitor.

[0199] The pharmaceutical composition in some embodiments contains the antibodies or antigen-binding fragments in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the antibodies or antigenbinding fragments, by multiple bolus administrations of the pharmaceutical composition, or by continuous infusion administration of the antibodies or antigen-binding fragments.Attorney Docket No: 205961-7120W01(00553)

[0200] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the antibodies or antigen binding fragments are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the antibodies or antigen binding fragments are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0201] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0202] Sterile injectable solutions can be prepared by incorporating the antibodies or antigen binding fragments in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0203] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.Attorney Docket No: 205961-7120W01(00553)

[0204] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by fdtration through sterile filtration membranes.

[0205] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0206] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0207] EXPERIMENTAL EXAMPLES

[0208] The invention is now described with reference to the following non-limiting Examples. The invention should not be construed to be limited solely to the compositions and methods described herein, but should be construed to include other compositions and methods as well These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein. One of skill in the art will know that other compositions and methods are available to perform the procedures described herein.Attorney Docket No: 205961-7120W01(00553)

[0209] Example 1: Generation of Humanized a2(I)Ct Antibodies

[0210] The variable region of the heavy chain (VH) was cloned into LB601 to express as a human chimeric heavy chain. The variable region of the light chain (VL) was cloned into LB603 to express as a human kappa chimeric light chain.

[0211] The heavy chain of the parental mouse anti-a2(I)Ct antibody was derived from the mouse germline, VH9-2. The light chain of the parental mouse anti-a2(I)Ct antibody was derived from the mouse germline, VK8-21. To humanize the parental mouse anti-a2(I)Ct antibody, the human germline VH1-2 or VH7-4-1 was chosen as an acceptor framework for CDR grafting of the human germline VH1-2 or VH7-4-1 heavy chain and the human germline VK4-1 was chosen as an acceptor framework for CDR grafting of the light chain. More specifically, Hl, H2 and H3 used VH7-4-1 and H4, H5 and H6 used VH1-2. Based on 3D modeling, six mutations were introduced into the VH1-1 or VH7-4-1 framework, resulting in 6 humanized a2(I)Ct heavy chain variants (H1-H6) that were cloned into LB602 for expression as a human IgG4 chimeric heavy chain. In addition, two mutations were introduced into the VK4-1 framework, resulting in 2 humanized a2(I)Ct light chain variants (L1-L2) that were cloned into LB604 for expression as a human heavy chain. Combining each of the H1-H6 variants with each of the L1-L2 variants results in the generation of 12 humanized anti-a2(I)Ct antibodies.

[0212] For producing the parental anti- a2(I)Ct antibodies, the parental LB601 heavy chain- and parental LB603 light chain expression constructs were transiently cotransfected in 293 cells. For producing the humanized anti- a2(I)Ct antibodies, pairwise combinations (12) of the humanized LB602 (H1-H6) and humanized LB604 (L1-L2) expression constructs were transiently cotransfected in 293 cells. Each of the 13 antibodies was purified by Protein A chromatography from 100 ml culture medium resulting in a yield of about 1-2 mg.

[0213] FIGs. 2A-2B are SDS-PAGE analyses of the antibodies under non-reducing (NR) to show the full antibody and under reducing (R) conditions to show the heavy and light chains of each antibody. FIG. 2A, lane 1 shows the parental anti-a2(I)Ct chimeric antibody (LB601 / 603). FIG. 2A, lanes 3-14 show each of the 6 heavy chain variants (H1-H6) in combination with the LI variant light chain. FIG. 2B, lanes 1-14 show each of the 6 heavy chain variants (H1-H6) in combination with the L2 variant light chain.

[0214] Antibody variants were further analyzed by size exclusion HPLC. All antibody variants showed similar elution profiles consistent with the molecular mass of an IgG-type antibody.Attorney Docket No: 205961-7120W01(00553)

[0215] Moreover, all variants showed a single elution peak, thereby indicating that they did not form any aggregates (chromatograms not shown).

[0216] Example 2; Binding Kinetics of Humanized a2(I)Ct Antibodies

[0217] The kinetics of association and dissociation of the 13 variants to a2(I)Ct was analyzed using an optical biosensor (OCTET Nl). For binding studies, human procollagen I was immobilized on the biosensor chip, and the pepsin-extracted mouse collagen I, which lacks the telopeptide regions, was used as a control. FIG. 3 shows the target molecules relevant to these studies. FIG. 3 A is a schematic of intact procollagen I. FIG. 3B shows collagen I that includes telopeptides; FIG. 3C shows collagen I in which the telopeptides were digested by pepsin.

[0218] In all binding assays, the highest concentration of the antibody variants added in solution was 300 nM. The lower concentrations represented a series of two-fold or three-fold dilutions of the starting concentration. Based on the kinetics of association and dissociation, the on and the off rates and KD values were calculated. Table 2 presents actual association / dissociation profiles together with fitted curves, the on rates and the off rates, the maximal response, and the KD values.

[0219] All the variants were found to bind intact human procollagen I molecules. None of the antibody variants interacted with pepsinized mouse collagen I lacking the telopeptide regions. The chimeric antibody LB601 / 603 exhibited an equilibrium dissociation constant (KD) of 8.52 nM, indicating a high binding affinity for human procollagen I. The binding kinetics of all the antibodies are summarized in Table 2.

[0220] Table 2. Procollagen I binding kinetics of humAbs.

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[0224] Example 3; Binding Specificity of the Humanized a2(I)Ct Antibodies

[0225] Although the biosensor-based binding assays demonstrated that all antibodies interact with native procollagen I, they could not show whether the procollagen I-antibody binding occurred via the epitope located within the pro-a2(I) chain.

[0226] To confirm that binding of the anti-a2(I)Ct variants to procollagen I is driven by the telopeptide regions, Western blot assays were performed in which procollagen I (with the C-terminal telopeptide (a2(I)Ct) and pepsin-digested procollagen I (without a2(I)Ct) chains were separated in polyacrylamide gels, electroblotted onto a nitrocellulose membrane and bound to the antibody variants. FIG. 3D shows a Coomassie blue-stained gel showing the procollagen I (Pro-I) and pepsin-digested Pro-I (i.e., collagen I, Col-I) chains (without telopeptides) separated in a polyacrylamide gel.

[0227] To show the interaction between the antibody variants and the pro-a2(I) chain of procollagen I, procollagen chains were separated in the gel, as shown in FIG. 3D. For this assay, denatured procollagen was loaded into one well stretching across the width of the gel. A molecular mass marker was loaded into a separate, regular width well. Following electrophoresis, the proteins were electroblotted onto a nitrocellulose membrane. Subsequently, the membrane was incubated in a blocking solution. Next, the membrane was placed into the MPX Blotting System (LICOR) that allows simultaneous assays of 24 different antibodies. The antibody variants were added to separate channels of the MPX device to interact with the procollagen chains for 2 h. Following a series of washes, antibody / procollagen chain complexes were detected with a specific “secondary” IRDye 800CW antibody (LI-COR). Based on the results shown in FIG. 4A, all antibody variants were found to specifically interact with the pro-a2(I) chain; interaction with a product of degradation of the pro-a2(I) chain was also apparent.Attorney Docket No: 205961-7120W01(00553)

[0228] To further show that the antibody variants did not bind to collagen chains lacking the telopeptide regions, a Western blot assay was carried out as described above, except instead of the pro-a2(I) chain, the pepsin-digested Col-I chains were separated on a polyacrylamide gel as shown in FIG. 4B. As shown in FIG. 4B, collagen chains in which telopeptides were removed with pepsin did not interact with analyzed antibody variants.

[0229] To demonstrate direct binding of the anti-a2(I)Ct antibodies to a2(I)Ct (telopeptide), streptavidin (2 pg / ml) was coated on ELISA plates. After blocking, biotinylated a2(I)Ct (0.1 pg / ml) was added to each well for 1 hour. After washing, a 2-fold dilution series of the anti-a2(I)Ct antibodies (starting at 2 pg / ml) were added. Absorbance readings (A450) were taken for detection of telopeptide-bound antibodies following addition of anti-human Fc HRP. FIG. 5A shows the results of this analysis for the anti-a2(I)Ct antibodies containing the LI light chain variant; FIG. 5B shows the results of this analysis for the anti-a2(I)Ct antibodies containing the L2 light chain variant. As shown in FIGs. 5A-5B, the parental antibody (LB601 / 603) bound telopeptide (a2Ct) with high affinity (EC50 about 0.1 pg / ml). Similar binding affinities were obtained when pairing H2, H3, H4, and H6 with LI (FIG. 3A) and when pairing H2, H3, H5, and H6 with L2 (FIG. 3B).

[0230] To further analyze the binding kinetics between the anti-a2(I)Ct antibodies and the a2(I)Ct telopeptide, binding of the a2(I)Ct telopeptide to the chimeric anti-a2(I)Ct antibody and the top 8 humanized anti-a2(I)Ct antibodies in FIGs. 5A-5B was examined by Octet binding analysis using the ForteBio Octet instrument. Briefly, 0.02 pg / mL of biotinylated a2(I)Ct, referred to as Bt-690, was loaded onto Streptavidin (SA) Octet sensors, and the loaded sensors were dipped into 7 serial dilutions of antibodies (300 nM start, 1:2 dilutions). Kinetic constants were calculated using a monovalent (1:1) binding model. The resulting binding curves in FIG. 6 were used to generate the following data in Table 3.

[0231] Table 3. Binding kinetics of humAbs with Bt-690 peptide.

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[0234] Attorney Docket No: 205961-7120W01(00553)

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[0237] In Table 3, “Full XA2” provides a measure of the goodness of curve fitting (not directly related to a parameter estimate). It is the sum of squared deviations, where deviation is the difference between the actual data point and the fitted curve. There is one value for each curvefit. Values close to zero indicate a good curve fit. “Full RA2” is the coefficient of determination (COD). It is an estimate of the goodness of the curve fit and is not directly related to the estimate of a specific parameter. Values close to 1.0 indicate a good curve fit.

[0238] LB602-H3 / 604-L2 exhibited the highest binding affinity, including a 2-fold better on rate compared to the parental antibody. It appears that avidity contributed to the apparent off rate which was beyond the instrument detection limit. LB602-H3 / 604-L2 and LB602-H6 / 604-L2 exhibited similar affinities (6-9 nM) as the parental chimeric antibody. LB602-H8 and LB602-H9 were further engineered from LB602-H3 and LB602-H6, respectively, to remove a potential deamidation site. The high antigen binding affinities of LB602-H8 / 604-L2 (H8L2) and LB602-H9 / 604-L2 (H9L2) were confirmed in a similar biosensor assay and determined to be 6.4 nM and 3.9 nM, respectively. The humanized AaA (AC A) H8L2 and H9L2 were used in the subsequent experiments.

[0239] Example 5; Inhibition of Collagen Fibril Formation by the Humanized H8L2 and H9L2 Variants

[0240] To show that the humanized H8L2 and H9L2 antibodies inhibit fibril formation, three different assays were carried out. In the first assay, human procollagen I was purified from cultures of dermal fibroblasts by ion-exchange chromatography and converted to collagen I using LysC enzyme at 25°C for 30 min. While digesting propeptides, cleavage with LysC preserves collagen telopeptides, i.e., the antibody’s epitope. At this stage, collagen molecules remain in non-aggregated, i.e., non-fibrillar form. Enzymatic activity of LysC was inhibited with Na-p-tosyl-l-lysine chloromethyl ketone (TPCK, Sigma-Aldrich) added to the final concentration of 1Attorney Docket No: 205961-7120W01(00553)

[0241] mM. Each of the humanized H8L2 and H9L2 antibodies, a human IgG control, and a no antibody control were separately added to the collagen monomers.

[0242] Collagen fibril formation was initiated by placing the samples into a heating block set to 37°C. The fibrils were formed for 24h. Fibril formation occurs due to the site-specific aggregation of collagen molecules as shown in FIG. 1. In the presence of antibodies that block these sites, aggregation into fibrils is not possible. Drops of incubated samples were transferred onto glass slides. The fibrils were observed under a light microscope in the dark field mode. In these settings, collagen fibrils, if formed, appear as needle-shaped aggregates or other fibrillar structures. If the fibrils go out of the focal plane, they appear as blurry structures. By this method, only fibrils are visible; collagen molecules that do not aggregate remain in solution and cannot be observed.

[0243] FIGs. 7A and 7C are representative images at 4x or lOx magnification, respectively, of collage fibrils formed in the presence of inactive IgG (hlgG) or the absence of antibody. FIGs.

[0244] 7B (4x magnification) and 7D (lOx magnification) are representative images showing the absence of visible fibrils in the presence of the humanized H8L2 and H9L2 antibodies, despite the presence of collagen molecules, indicating a lack of collagen aggregation into fibrils.

[0245] In a second assay, parallel samples were analyzed by electrophoresis. Briefly, following fibril formation, the samples were centrifuged for 30 min. Since collagen fibrils are large aggregates, they settle at the tube’s bottom as pellets (P). Non-aggregated collagen molecules remain in supernate (S). Following centrifugation, P and S fractions are loaded onto a gel.

[0246] Subsequently, collagen from dissociated fibrils (P) and non-aggregated molecules (S) is visualized by staining with Coomassie blue dye. The results of this analysis are shown in FIG. 8. The results indicate that in the presence of blocking antibodies, almost all collagen molecules remain in nonaggregated, i.e., non-fibrillar form. In contrast, the fibrillar fraction is readily visible in the presence of noninhibitory hlgG or the absence of an antibody. It is noted that some monomers always remain in non-aggregated form, even without any antibody. This is to maintain the critical concentration of collagen molecules to allow their aggregation. The fibril formation will not continue if the collagen concentration falls below this critical concentration (e. ., due to the trapping of collagen molecules in the fibrils).

[0247] In the third assay, human procollagen I was similarly purified from cultures of dermal fibroblasts by ion-exchange chromatography and converted to collagen I using LysC enzyme atAttorney Docket No: 205961-7120W01(00553)

[0248] 25°C for 30 min. Enzymatic activity of LysC was inhibited with TPCK. Each of the humanized H8L2 and H9L2 antibodies and a human IgG control were separately added to the collagen monomers. In each sample, the antibodies were added @ 5 concentrations: 270 nM, 90 nM, 30 nM, 10 nM, and 3 nM. The concentration of procollagen was constant @ 240 nM. Thus, the molar collagen: antibody ratios were —1:1, 1:0.3, 1:0.1, 1:0.03, and 1:0.01. Each of the humanized H8L2 and H9L2 antibodies and a human IgG control were separately added to the collagen monomers. In each sample, the antibodies were added @ 5 concentrations: 270 nM, 90 nM, 30 nM, 10 nM, and 3 nM. The concentration of procollagen was constant @ 240 nM. Thus, the molar collagen:antibody ratios were —1:1, 1:0.3, 1:0.1, 1:0.03, and 1:0.01.

[0249] Collagen fibril formation was initiated by placing the samples into a heating block set to 37°C. The fibrils were formed for 24h. Following fibril formation, the samples were centrifuged for 30 min. Since collagen fibrils are large aggregates, they settle at the tube’s bottom as pellets (P). Non-aggregated collagen molecules remain in the supemate (S).

[0250] Following centrifugation, P and S fractions were loaded onto a gel. Subsequently, collagen from dissociated fibrils (P) and non-aggregated molecules (S) was visualized by staining with Coomassie blue dye. The gels were scanned, and the collagen bands were quantified by measuring signal intensities (FIG. 9A). Then, the P signal :S signal ratios were calculated for each sample. These ratios were plotted against the concentration of an antibody (FIG. 9B). High P / S ratios indicate that most collagen is in the pellet (P), / .c'., fibrils. In contrast, small P / S ratios indicate that most collagen molecules remain as non-aggregated monomers in the supemates (S).

[0251] The results in FIG. 9B indicate concentration-dependent inhibition of collagen fibril formation by the H8L2 and H9L2 variants. Under similar conditions, the inactive human IgG control (hlgG) did not inhibit the formation of fibrils (FIG. 9B)

[0252] Example 6: Binding Kinetics of Humanized Dual-Targeted Anti-a2Ct / Collagen Antibodies to Human Procollagen

[0253] Humanized dual-targeted anti-a2Ct / collagen antibodies were prepared in which the humanized anti-a2Ct antibodies of the present invention were modified by fusing a collagen-binding peptide (TKKTLRT, SEQ ID NO: 43) to the C-terminus of the antibody or at positions before the hinge or after the hinge of the antibody to minimize the potential effects on antigenAttorney Docket No: 205961-7120W01(00553)

[0254] binding. Inclusion of the collagen-binding peptide (CBP) was designed to target the anti-a2Ct antibodies to regions where excess collagen is present.

[0255] Three antibody variants were constructed. Each of these variants carries a CBP flanked by the spacer, GGGGS (SEQ ID NO: 57) to form a ggggsTKKTLRTggggs CBP (SEQ ID NO: 60). LB991 is a heavy chain variant of LB602-H9 comprising the amino acid of SEQ ID NO: 66, which contains the CBP peptide of SEQ ID NO: 62 before (on the N-terminal side) the IgG4 heavy chain hinge region. LB992 is a heavy chain variant of LB602-H9 comprising the amino acid of SEQ ID NO: 68, which contains the CBP peptide of SEQ ID NO: 60 after (on the C-terminal side) the IgG4 heavy chain hinge region. LB993 is a light chain variant of LB604-L2 comprising the amino acid of SEQ ID NO: 70, which contains the CBP peptide of SEQ ID NO: 61 at the C-terminal end ofLB604.

[0256] In variant H9 (used in LB991 and LB992) the sequence NT in HCDR2, a site of known potential deamidation liability was modified so that the N was changed to an S resulting in ST which is no longer subject to this liability. LB991 and LB992 which carry VH H9 is derived from H6 with the indicated N->S mutation. Variant dual-targeted anti-oc2Ct / collagen antibodies were produced by separately cotransfecting cells with LB 991 or LB992 in combination with LB604-L2 or cotransfecting cells with LB993 in combination with LB602-H9.

[0257] A biosensor (OCTET Nl) was utilized to measure kinetics of binding between five different anti-a2(I)Ct antibodies and six different collagen-based targets (Table 3). The anti-a2(I)Ct antibodies included ACA and LB602 (humanized anti-a2(I)Ct variants, no CBP) and LB991, LB992, and LB993 variants containing CBPs. The collagen-binding targets included Pro-I, Pro-I-peps, tendon collagen I, mouse collagen I, chick collagen II, and C2-telopeptide (Table 3).

[0258] Pro-I corresponds to native procollagen I (FIG. 3A) produced from cultured dermal fibroblasts, which includes procollagen propeptides (NP, CP), telopeptides (Nt, Ct), and the triple-helical domain (TH). LB991, LB992, and LB993 variants containing CBPs were expected to bind Pro-I via a2(I)Ct and TH (Table 3). In contrast, ACA and LB602 were expected to bind via the a2(I)Ct domain only.

[0259] Pro-I-peps corresponds to native procollagen I digested with pepsin. Pepsin digests globular propeptides and parts of epitope-containing telopeptides (FIG. 3C); TH remains intact. Pro-I-peps was expected to bind LB991, LB992, and LB993 variants containing CBPs viaAttorney Docket No: 205961-7120W01(00553)

[0260] a2(I)Ct (if incompletely digested) and TH. Tendon collagen I (predominantly collagen I) was isolated from rabbit tendon using pepsin-based extraction and was similarly expected to bind antibody variants via a2(I)Ct (if incompletely digested) and TH. Mouse collagen I (predominantly collagen I) was isolated from mouse skin using acetic acid extraction (no pepsin). Previous results showed mouse a2(I)Ct has poor binding affinity for the parental anti- a2(I)Ct antibody. Hence, this collagen was expected to bind the antibodies predominantly via TH-CBP interactions. Chick collagen II is a commercially derived collagen isolated from the chick sternum. This collagen type was expected to bind the antibodies predominantly via TH- CBP interactions. C2-telopeptide is a synthetic anti-a2(I)Ct antibody target expected to bind a2Ct-binding sites of the antibodies.

[0261] Briefly, a collagen-based target was biotinylated and immobilized on an avidin-coated sensor. After determining the concentration of an antibody variant, serial dilutions of the variant ranging from 6.25 to 400 nM were prepared. Subsequently, ko» and k measurements were recorded for each antibody concentration and a KD value was calculated using the global fitting approach. Binding kinetics for each antibody variant / collagen target pair were measured (Table 3).

[0262] Table 3. Binding Kinetics of Dual-Targeted Anti-a2(I)Ct / Collagen Antibodies (LB 991, LB992, and LB993) and Anti-a2(I)Ct variants (ACA and LB602)

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[0265] Attorney Docket No: 205961-7120W01(00553)

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[0268] As shown in Table 3, all antibody variants exhibited a high affinity for binding collagen variants with the a2(I)Ct target. Antibody variants with CBP domains showed significantly higher affinities for binding collagen targets without the canonical a2(I)Ct epitope (i.e., mouse collagen I, chick collagen II, and Pro-I-peps, tendon collagen). These results indicate that the CBP domains embedded into the antibody’s structure provide an additional collagen-binding mechanism. Despite certain differences in kinetics parameters, all three CBP-containing variants appear to have similar collagen binding properties.

[0269] Example 7; Generation of Production Cells for Anti-a2(I)Ct Humanized Antibodies Production cells expressing the leading anti-human a2(I)Ct antibodies, LB602- H8 / LB604-L2 (H8L2) and LB602-H9 / LB603-L2 (H9L2) were generated with CHO-S cells.

[0270] The CHO-S cells were originally purchased from Invitrogen on 1 / 26 / 10 (P / N 51-4449, Lot# 1102005) and were initially maintained in Freestyle CHO media (Invitrogen) under serum- free condition. After thawing, the cells were cultured in Freestyle CHO media supplemented with GlutaMax (4mM). After 5 passages, the cells were frozen in Freestyle CHO media supplement with 10% DMSO (Sigma) at 1 x 107cells / ml. The CHO-S cells were subsequently maintained in CD FortiCHO media (Invitrogen) under serum -free condition, supplemented with 4 mM GlutaMax (Invitrogen). To start a culture, frozen cells were rapidly thawed at 37°C and diluted in 10 ml of pre-warmed growth media. The cells were then spun down, resuspended in 30 ml of the growth media, and placed in an orbital shaker at 120 rpm, 37°C, and 5% CO2. The cells were passaged every 3 or 4 days and kept between 0.2 - 3 x 106cells / ml.

[0271] A proprietary SwiMR expression system (Panorama / Larix Biosciences) was employed for the facile development of antibody production cell lines, utilizing a switchable membrane reporter to facilitate the isolation of highly productive cells via FACS. The antibody heavy and light chains are expressed under the control of constitutive human EFla promoters in two separate vectors. An internal ribosome entry site (IRES)-mediated bicistronic expression cassette of membrane-anchored GFP is present downstream of the gene of interest (GOI), e.g., antihuman a2(I)Ct heavy chain (FIG. 10A). The IRES-GFP cassette is flanked by LoxP sites for later removal from the chromosome. The GFP expression level is used to mark the expressionAttorney Docket No: 205961-7120W01(00553)

[0272] level of the antibody. The high producing cells were isolated by FACS and in which the GFP cassette can be removed from the chromosome by treatment with recombinant Cre DNA recombinase.

[0273] H8L2 and H9L2 were expressed as engineered human IgG4 antibodies that are stable with minimal effector functions. The heavy chains H8 and H9 with their corresponding signal peptide domains were cloned into the SwiMR expression vector LB602 containing an engineered Fc region of human IgG4 (S228P / F234A / L235A) under the control of a human EFla promoter for strong and constitutive mammalian cell expression. The IRES-GFP cassette is positioned downstream of the heavy chain and is flanked by two LoxP sites. The vector LB602 carries a puromycin resistant gene for mammalian selection and an ampicillin gene for bacteria propagation. The light chain L2, including the signal peptides, was cloned into the SwiMR expression vector LB604, which carries a neomycin-resistant gene for mammalian selection and an ampicillin gene for bacteria propagation. The plasmid maps are shown in FIGs. 9B and 9C.

[0274] CHO-S cells were maintained in CD FortiCHO media supplemented with 4 mM GlutaMax between 0.2 - 3 x 106cells / ml in shaking flask (120 rpm, 37°C, 5% CO2). Following the manufacturer's recommendation, transfection was performed with Freestyle Max regent (Invitrogen). After linearized with restriction enzyme Sea I, LB602 and LB604 plasmids were transfected into 100 ml of CHO cells with Freestyle Max transfection reagent (Invitrogen) and was selected with 10 pg / ml of puromycin and 500 pg / ml of G418 for 2 weeks.

[0275] After stable selection, the top ~1% of the cells with the highest GFP signal were sorted out as a pool containing 200,000 cells (FIG. 10). The cells were pelleted and resuspended in Freestyle CHO media before sorting. The sorting was performed in Stanford Shared FACS Facility. The cells were sorted into the regular CD FortiCHO growth media and then cultured in CD FortiCHO media supplemented with 10 pg / ml of puromycin, 4 mM GlutaMax, and antidumping agent (1 : 1000). The sorted CHO cells were designated as 44.42.2bp l for H8L2 (FIG.

[0276] 10A) and 44.42.3bpl for H9L2 (FIG. 10B) and were used for antibody production to support pre-clinical in vitro and in vivo studies.

[0277] For GMP manufacturing to support clinical applications, full cell line development is continued with the sorted pools. Typically, the pools are sorted again for the top 1% of the highest GFP signals and subjected to treatment of recombinant Cre to remove the GFP cassette. The GFP negative cells are then single-cell sorted into 384-w plates for cloning. The clones areAttorney Docket No: 205961-7120W01(00553)

[0278] screened for the antibody production in 96-w, 24-w, and 6-w plates, and eventually in shaking cultures. The top clones are further characterized for sequence integrity, the production level, and stability.

[0279] 8: Retention of the anti a2(I)Ct (ACA) antibodies containing collagen-binding

[0280]

[0281] within collagen-rich matrices formed in cell culture conditions Retention of the humanized anti-collagen I antibody (H-ACA) and its modified variant containing collagen-binding peptide (CBP-ACA) in collagen-rich matrices formed by cultured fibroblasts was investigated. Briefly, fibroblasts were cultured for 10 days to develop a collagen-rich matrix (FIG. 12A). Subsequently, the humanized ACA (H-ACA) or H-ACA with CBP (CBP-ACA) variants were added to fibroblast cultures at 200 pg / ml. Finally, the cell layers were washed extensively to remove unbound antibodies. Next, the cell layers were lysed using a lysis buffer to check the presence of the antibodies trapped within collagen-rich matrices.

[0282] Subsequently, proteins present in the lysates were separated by electrophoresis. The presence of the antibodies was analyzed by western blot using anti-human primary antibodies conjugated with a green chromophore. Pixel intensities of the antibody-specific heavy (Hy) and light (LK) chains were measured by densitometry. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was measured as an internal protein-loading control. GAPDH was detected using an anti-GAPDH antibody conjugated with a red chromophore.

[0283] As shown in FIG. 12B, the amount of CBP-modified H-ACA (CBP-ACA) present in the collagen-rich matrix was found to be 3-fold higher than its unmodified counterpart. As determined during earlier studies (Fertala, J. et al., Connect Tissue Res 2014 Apr;55(2): 115-22), the original ACA (with no CBP) has a high binding affinity for free collagen molecules (those not incorporated into fibrils). In contrast, it binds poorly already-established fibrils. Hence, the result in FIG. 12B showing improved retention of CBP-ACA within collagenous matrices formed in cell layers indicates that the CBP domain mediates the retention of the CBP-ACA within the collagen-rich matrices.

[0284] 9: Rabbit model of arthrofibrosis

[0285] The effects of the modified anti-collagen antibody (mACA) on collagen-based deposits formed in the posterior knee capsules was evaluated in a rabbit arthrofibrosis model. This rabbitAttorney Docket No: 205961-7120W01(00553)

[0286] model meets essential criteria for a valid preclinical animal model. First, the rabbit musculoskeletal system shares fundamental features with that of humans, including the composition and structure of bones, ligaments, tendons, and muscles. Additionally, rabbit and human collagenous proteins exhibit similar patterns of biosynthesis and fibrillogenesis. Notably, the therapeutic target in this study, the C-terminal telopeptide of the a2(I) chain, is identical (100%) between rabbits and humans.

[0287] Methods

[0288] Five 8- to 12-month-old New Zealand White (NZW) rabbits (3M + 2F)(Charles River, Wilmington, MA) were employed to study the impact of modified anti-collagen antibody (mACA) on key parameters characterizing collagen-rich deposits formed in a model of post-traumatic arthrofibrosis [1-6],

[0289] Post-traumatic contracture was generated by simulating an intra-articular fracture and bleeding (using drill holes) and a capsular injury (with a hyperextension injury) in the right knee and maintaining it in a flexed position using a Kirschner wire (K-wire) for 8 weeks. The contralateral uninjured limb served as the uninjured control. A refillable pump (see below) was installed subcutaneously to deliver tested mACA directly into the articular cavity of the injured knee joints. In brief, an incision in the middle of the right knee was made starting 5 mm proximally to the patella and extending distally to the tibial tubercle and down the tibia.

[0290] Subsequently, a lateral parapatellar arthrotomy was made, and the patella was dislocated medially. Following flexing the knee, the fat pad was released and retracted medially. Next, the anterior and posterior cruciate ligaments (ACL and PCL) were transected, and the knee was hyperextended to 45° to disrupt the posterior capsule (PC). Employing a 2-mm drill, a hole was made in the medial femoral condyle. Beginning as far posteriorly in the notch as possible, and exiting out through the lateral condyle, a second 2-mm hole was made. Starting from the notch, a custom-made silicone catheter (0.6 mm ID / 1.2 mm OD, Access Technologies, Skokie, IL) with a fixed retention bead at one end was passed through the hole. While the retention bead secured the catheter inside the operated knee, the opposite end was connected to a pump.

[0291] The injured knee was fixed in a flexed position by employing a K-wire. In brief, a drill was driven in the anterior-posterior direction to create a 2-mm bicortical drill hole in the tibia. Subsequently, a 1.6-mm K-wire (Modern Grinding, Port Washington, WI) was passed through the hole in the tibia (FIG. 13). Flexing the knee, the K-wire was maneuvered to exit through theAttorney Docket No: 205961-7120W01(00553)

[0292] femur's lateral side incision. Two 90° bends, approximately 1 cm apart, were made to form a hook at the end of the K-wire. Next, the hook was positioned around the femur (FIG. 13).

[0293] Finally, the knee was fixed at flexion by tightening a nut on the opposite, threaded end of the K-wire.

[0294] Pump installation. Utilizing a subcutaneous, programmable pump (iPrecio SMP-200, Primetech Corp., Tokyo, Japan) has allowed us to control mACA delivery directly into the knee injury site. The pump was placed into a subdermal pocket created during surgery (see above). Employing a stainless-steel tube connector (Access Technologies, Skokie, IL), the free end of the catheter was connected to a silicon tube attached directly to the refillable pump (FIG. 13), which was filled with 0.9 ml of mACA at a concentration of 30 mg / ml. Nylon sutures (Ethicon Inc., Somerville, NJ) were tied around the silicon tubes at their juncture to ensure the stability of the connection site. Finally, the incisions were closed with 4-0 absorbable sutures (Redisorb, MYCO Medical, Cary, NC). Following the surgery, the rabbits were allowed unrestricted cage activity.

[0295] K-wire removal. After 8 weeks of immobilization, the rabbits underwent a second surgery to remove the K-wires. Bridging heterotopic ossification formed along the K-wire was manually disrupted. Subsequently, the rabbits were allowed 4 weeks of unrestricted cage time, after which they were euthanized.

[0296] Antibody delivery. In addition to the initial mACA portion supplied by the pump implanted during the initial surgery (see above), 4 weeks later, the pump was refilled with 0.9 ml of mACA at a concentration of 30 mg / ml.

[0297] Blood collection and analysis. Blood samples were collected before and 4, 8, and 12 weeks after the initial surgery. Complete blood counts (CBCs) were performed using the Element HT5 instrument (Antech Diagnostics, Inc., Levittown, PA).

[0298] Histology’ of tissues and organs. At euthanasia, various organs and tissues (eg., brain, heart, liver, tendons, nerves) were collected, processed for histology, and stained with H&E to assess structural and inflammatory changes that may have been caused by mACA [6], Mechanical assays of the flexion contracture. Joint immobilization and knee injury can lead to stiffness and flexion contracture, primarily due to scarring of the posterior capsule (PC). Thus, measuring contracture provides a clinically relevant endpoint to assess mACA efficacy in modulating collagen fibrillogenesis [7, 8],Attorney Docket No: 205961-7120W01(00553)

[0299] The flexion contracture assays were done using a custom-made instrument (FIG. 14) [2, 6, 8], In brief, the injured or the contralateral uninjured leg was fixed in the instrument with the tibia and femur positioned at a right angle. This position was considered the starting point of zero degrees. Subsequently, applying the 40° / min loading rate, an extension torque of 0.2 Nm was applied. Upon reaching the value of 0.2 Nm, the angle corresponding to the joint extension was recorded. The injured knee flexion contracture was then expressed as the ratio of the uninjured control knee extension to the injured contralateral knee extension [2, 6-8], Hence, relatively low ratio values indicated relatively small flexion contracture, while large ratio values indicated more severe flexion contracture.

[0300] Assays of PCs. Since PC scarring drives knee stiffness, PCs were dissected for histological, biochemical, and spectroscopic assays focused on collagenous material (see below) [6-9],

[0301] Histological assays of collagen fibrils. Histological sections of the PCs were stained with hematoxylin and eosin (H&E) to visualize gross tissue morphology and cellularity. In addition, PC sections were stained with collagen-specific picrosirius red dye (FIG. 15). Combining this staining with polarized-light microscopy enabled an approximation of the fibrils' thickness, organization, and packing density [7, 10-12], Studies verified that as the thickness and packing of fibers increase, their birefringence color changes in the polarized light from green to yellow to orange to red, / .<?., from shorter to longer wavelengths [10, 13-16],

[0302] Employing a polarizing microscope (Eclipse LV100POL, Nikon Inc., Melville, NY) and the NIS Elements software (Nikon Inc.), the following groups of the birefringence colors were defined in captured images: (i) green birefringence (GB, identifies thin, loosely packed fibrils), (ii) yellow birefringence (YB, identifies intermediate-thickness fibrils), and (iii) red birefringence (RB, identifies thick, tightly packed fibrils) (FIGs. 15B-15C) [6-8],

[0303] All fibrils in captured viewing areas were analyzed [7, 17], In brief, the above birefringence colors were defined by applying the software’s “color threshold” function.

[0304] Subsequently, the software determined the areas occupied by pixels corresponding to the defined colors. Considering the sum of all pixels to be 100%, the percentage of each color group in the analyzed samples was calculated. The exact threshold settings were applied to all images to ensure consistency in defining birefringence colors. Automated calculations of all fibrils in the viewing areas eliminated any potential bias.Attorney Docket No: 205961-7120W01(00553)

[0305] Fourier transform infrared spectroscopy (FTIR) -based assays of the collagen content and cross-links. FTIR spectroscopy of tissue samples provides information about their composition and the spatial distribution

[0018] , Here, this method was used to measure the relative collagen content in the scar tissues formed in the PCs of the mACA-treated rabbits. As a non-collagenous reference, a peak corresponding to sulfated glycosaminoglycans (GAGs), a common non-collagenous component of connective tissues, was selected. The GAG / collagen ratios were calculated based on the areas of the spectral peak corresponding to the sulphated GAGs, centered around 1064 cm’1wavenumber (v), and the spectral peak corresponding to collagen, centered around 1338 cm’1v [6, 19, 20], Please note that the relation between the ratio values and a relative collagen amount is negative.

[0306] In addition, the maturity of collagen cross-links was analyzed by measuring the pyridinoline (PYR) peak / dehydro-dihydroxynorleucine (de-DHLNL) peak ratios [6, 21, 22], Hence, the maturity of collagen fibrils was defined as the relative content of the “mature” trivalent cross-links (PYR) vs. the “immature” divalent PYR precursor cross-links (de-DHLNL).

[0307] Since the PYR / de-DHLNL ratio often increases during the fibrotic process, its assays provide a relevant parameter to evaluate the fibrotic status of collagen-rich deposits formed in the presence of the mACA [23-25], The mature trivalent PYR cross-link centers around 1660 cm’1v, and the immature divalent deDHLNL cross-link centers around 1690 cm’1v.

[0308] Paraffin-embedded 5-pm-thick tissue sections were deposited on the MirrIR low-e microscope slides (Kevley Technologies, Chesterland, OH). An FTIR spectrometer (Spotlight 400, Perkin Elmer, Waltham, MA) was used to analyze the regions of interest (ROIs) corresponding to the PC collagen-rich areas (FIG. 15). The measurements were done in the 4000 cm’1to 748 cm’1v spectral range, at a pixel resolution of 50 pm, 8 scans per pixel, and a spectral resolution of 4 cm’1v. The Spectrum Image software generated co-added spectra from scanned ROIs (PerkinElmer, Inc ).

[0309] In all assays of the FTIR-derived spectra, overlapping peaks were deconvoluted and analyzed based on the second-order derivative spectra and pre-determined bell-type Gaussian peak fitting function using the OriginLab software (version 2025, OriginLab Corporation, Northampton, MA, USA) [6, 26, 27],

[0310] Total collagen content. To analyze the impact of mACA on collagen content in scar tissue formed in the PCs, collagen-specific hydroxyproline (HP) was measured. In brief, PCAttorney Docket No: 205961-7120W01(00553)

[0311] samples were frozen in liquid nitrogen and then pulverized in a stainless-steel mortar. Next, the samples were treated with a 3: 1 mixture of chloroform and methanol to extract the lipids, then lyophilized, and weighed. Then, tissues were hydrolyzed in 6 N HC1. Subsequently, the HP content was measured using an HP kit (Sigma-Aldrich, St Louis, MO) [7, 28], The HP content was used to calculate the collagen content / tissue dry mass.

[0312] Collagen III: collagen I ratio. PC portions were also used to measure collagen Ill-to-collagen I ratios. The pepsin-soluble collagen fraction was obtained by digesting the samples with porcine pepsin (Sigma-Aldrich) prepared in 0.5 M acetic acid at a final concentration of 1 mg / ml. Following extraction, collagen was precipitated with 1.2 M NaCl. Subsequently, collagen pellets were washed with cold water and resuspended in 0.5 M acetic acid. To separate the monomeric forms of collagen I chains from collagen III monomers, an interrupted electrophoresis method was employed that takes advantage of the fact that, unlike the collagen I a-chains, the collagen III a-chains are bound together via reducible disulfide bonds

[0029] , In brief, heat-denatured collagen samples were initially run in a 6% polyacrylamide gel in non-reducing conditions. After 15 minutes of running the electrophoresis, a 10 pl aliquot of 0.5 M dithiothreitol (DTT) was added to each well, and electrophoresis was continued. Subsequently, pixel intensities of Coomassie blue-stained protein bands corresponding to the al (III) and the combined al (I) + a2(I) chains were measured by densitometry (Image Studio Lite, LLCOR Biotechnology, Lincoln, NE), and then collagen III: collagen I ratios were calculated (FIG. 16) [7].

[0313] Because the initial measurements of collagen I and collagen III extracted from the PCs of ACA-treated and control rabbits were obtained using now-outdated densitometry software, those measurements were reanalyzed them (together with new gels) using archived digital images of stained gels using Image Studio Lite software.

[0314] Data analysis. The primary goal of these studies was to engineer mACA and characterize its binding affinity and specificity for interaction with the native procollagen I target. A secondary, exploratory element of the study was testing the mACA utility to reduce post-traumatic scarring in vivo.

[0315] Due to the exploratory nature of the in vivo studies, relevant results (obtained in a blinded fashion) are presented in graphic form, including the means and standard deviations. The results obtained in mACA-treated rabbits were compared with corresponding historical data fromAttorney Docket No: 205961-7120W01(00553)

[0316] identical models, including non-modified ACA (ACA)-treated and non-treated groups [5, 6, 8, 30],

[0317] Results

[0318] Initially, five NZW rabbits (three males and two females) were employed to evaluate the mACA's impact on post-traumatic scar formation in the arthrofibrosis model. Following the initial surgery, one male rabbit was sacrificed and excluded from the study due to failure of the K-wire. All remaining rabbits were maintained for 12 weeks and are included in the study.

[0319] Blood collection and analysis. CBC provides valuable information about potential pathological changes that could have occurred due to the mACA application. (FIG. 17) Moreover, Tables 4A and 4B indicate that all cellular parameters stayed within their respective rabbit-specific physiological ranges. These results are consistent with the CBC assays performed earlier in rabbits treated with ACA [6], Consequently, CBC results obtained here strongly indicate that mACA does not alter normal blood cell parameters, thereby suggesting the safety of this ACA variant.

[0320] Table 4A. CBC in the mACA-treated rabbits.

[0321] >

[0322] "

[0323]

[0324] WBC, white blood cells; NEU, neutrophils; LYM, lymphocytes; MONO, monocytes; EOS, eosinophils; BAS, basophils; NEU %, percentage of neutrophils; LYM %, percentage of lymphocytes; MONO %, percentage of monocytes; EOS %; percentage of eosinophilsAttorney Docket No: 205961-7120W01(00553)

[0325] Table 4B. CBC in the mACA-treated rabbits.

[0326]

[0327] BAS %, percentage of basophils; HCT, hematocrit; HGB, hemoglobin; RBC, red blood cells; MCV, mean corpuscular volume; RDW %, red blood cell distribution width; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet count; MPV, mean platelet volume.

[0328] Histology of tissues and organs. Although mACA was applied locally, due to diffusion into the bloodstream, this antibody may impact distant anatomical sites. Thus, histological evaluation of tissues and organs provides valuable initial information about the safety of a tested biologic. Despite long-term treatment, it was demonstrated that mACA does not cause unwanted changes in crucial tissues and organs, as evidenced by the lack of structural abnormalities or excess inflammatory cells (FIGs. 18A-18E). In particular, the lack of mACA effects on collagen I-rich tissues, including tendons, sciatic nerves, muscles, lungs, and others, indicates the safety of this antibody. Consequently, modifications introduced to the mACA do not alter its safety profile compared to the parent ACA variant, whose safety has already been established in earlier studies [6].Attorney Docket No: 205961-7120W01(00553)

[0329] Mechanical assays of the flexion contracture. Applying the rate of loading set to 40° / min, an extension torque of 0.2 Nm was applied to the femur. Upon reaching the value of 0.2 Nm, the angle corresponding to the joint extension was recorded, and the femur was moved to the starting position at the same rate (FIGs. 14, 19). The injured knee flexion contracture was expressed as the ratio of the uninjured knee extension to the injured contralateral knee extension [6], Hence, relatively low ratio values indicated mild flexion contracture, while large ratio values indicated severe flexion contracture.

[0330] Comparing the mACA-treated rabbits to historical data from the ACA-treated and CTR rabbits indicates a marked decrease in the flexion contracture in the mACA and ACA groups vs. CTR (FIG. 20) [6], Exploratory results also show that the mean value of the flexion contracture in the mACA group was smaller than in the ACA group.

[0331] Histological assays of collagen fibrils in PCs. Collagen I-based fibrils, their structure (i.e., diameter, lateral aggregation, and packing), and cross-linking define the mechanical properties (e.g., stiffness) and stability (e.g., resistance to proteolytic degradation) of scar tissue. Consequently, all anti-fibrotic approaches aim to decrease the amount of collagen I by blocking its biosynthesis steps or targeting the proliferation of cells that produce this protein. In contrast, the present approach blocks extracellular collagen fibrillogenesis using ACA [6, 8, 31], Consequently, measuring collagen fibrils in tissues targeted by ACA or its modified variant mACA provides a valid parameter to determine the anti-fibrotic efficacy of these biologies.

[0332] Picrosirius red-staining of collagen fibrils, combined with polarized-light microscopy, allows for the quantification of collagen fibril sub-populations that differ in diameter and packing density. This has been observed that during wound healing and scar formation, the relative content of thin, green-birefringence fibrils increases in injured PCs [6-8], Consistent with the dynamics of collagen fibril formation in vivo, this increase was interpreted as the de novo formation of thin neo-fibrils during PC repair by scarring. Indeed, collagen fibrils first form as thin microfibrils that gradually aggregate laterally to form thicker fibrils and, finally, thick fibers. In one example, Frank et al. demonstrated the formation of small-diameter fibrils for the first 40 weeks of healing the injured rabbit medial collateral ligament

[0032] , After that time, the fibril diameter gradually increases due to fibril aggregation and collagen turnover. Still, two years after injury, the fibrils did not reach the diameters typical for uninjured ligaments.Attorney Docket No: 205961-7120W01(00553)

[0333] Results presented here indicate that compared to uninjured PCs, the relative amount of thin, green-birefringence fibrils in injured PCs increases in all groups. Nevertheless, the increase in the mACA group (approximately 16%) studied here, and the historical AC A group (approximately 26%), was markedly lower (FIG. 21) [6], Without wishing to be bound by any theory, it is believed that this relatively minor increase in the antibody-treated groups was due to blocking collagen aggregation at various stages of fibrillogenesis.

[0334] Fourier transform infrared (FTIR) spectroscopy-based assays of the collagen content and cross-links. As indicated earlier, the ultimate goal of all anti-fibrotic approaches is to reduce the amount of collagen in excessive scars responsible for fibrotic tissue malfunction. Hence, measuring the collagen content in target tissues is a crucial method for assessing the progress of fibrosis and the effectiveness of a tested anti-fibrotic therapeutic. Moreover, research demonstrated that the fibrotic process is propagated by scar stiffness, and this parameter increases with increased cross-linking of collagen fibrils. Consequently, reducing collagen crosslinks is a logical anti-fibrotic goal

[0033] ,

[0335] FTIR spectroscopy provides a valuable method for assaying many connective tissues, including PCs [6, 18], and was employed to measure the relative collagen content and status of mature, trivalent cross-links in mACA-treated PCs from injured legs and compare the results to historical data from ACA-treated and CTR rabbits (FIGs. 22A-22D)[6],

[0336] As indicated in FIG. 22C, the GAG: collagen ratios have increased in the mACA-treated and ACA-treated groups, indicating that the relative collagen content has decreased markedly in the antibody -treated groups, compared to the control. Based on earlier studies, it was postulated that this decrease in the relative collagen content is due to antibody-dependent inhibition of collagen fibrillogenesis. In support of this notion are studies that demonstrated that while fibrilincorporated collagen molecules are relatively stable, free collagen molecules are unstable in vivo, thereby prone to degradation in the tissue environment [6, 34],

[0337] As demonstrated by the decrease of the PYR / deDHLNL ratio in FIG. 22D, the relative content of trivalent PYR cross-links has decreased notably in the PCs isolated from the injured legs of the antibody-treated groups, compared to the control. Also, this decrease was markedly larger in the mACA-treated group than in the ACA-treated group.

[0338] It is postulated that this possible decrease could result from the antibody-mediated blocking of collagen cross-linking formation and maturation. Further, it is believed that antibodyAttorney Docket No: 205961-7120W01(00553)

[0339] binding to the C-terminal telopeptide region, a crucial site for lysine-dependent cross-link formation, may prevent the lateral aggregation of newly assembled thin fibrils and hinder vital interactions between potential cross-linking sites [35, 36], Altering collagen cross-linking represents a novel anti-fibrotic action of ACA.

[0340] Still, the possibility that the decrease of the PYR / deDHLNL ratio resulted partly from the increased amount of new thin fibrils (green-birefringence fibrils in FIG. 21) produced in response to injury cannot be excluded. It is expected that in newly synthesized fibrils, immature divalent cross-links are the dominant form.

[0341] Total collagen content. While the microscopic and spectroscopic tissue assays provide information on the relative collagen content, measuring collagen-specific hydroxyproline (HP) in acid-hydrolyzed tissue offers information about the absolute collagen content per tissue mass unit. FIG. 23 presents the results of assays on collagen content in PCs from the mACA-treated rabbits, comparing them with historical data from ACA-treated and control rabbits [8], The results indicate that in all groups, the collagen content measured 12 weeks after injury in the injured PCs tends to decrease compared to uninjured PCs (FIG. 23 A). This somewhat counterintuitive result is interpreted by the fact that 12 weeks after injury, the injured PCs' healing / remodeling process is not fully completed. In support of this notion are studies demonstrating continuous remodeling of collagen fibrils 2 years after medial collateral ligament injury in a rabbit model

[0032] ,

[0342] As indicated by the uninjured PC / injured PC collagen content ratios, the most prominent decrease in the collagen content in the injured PCs was observed in the mACA-treated rabbits. In contrast, the amount of collagen content in the PCs from the ACA-treated rabbits was not significantly different from that of the control (FIG. 23B). These results suggest that, compared to ACA, mACA has greater potential to reduce the collagen content in healing PCs. This would suggest that the multiple mACA-binding sites in collagen I (note that collagen I binds ACA only at one site) block collagen fibrillogenesis (i.e., nucleation of collagen fibril formation and aggregation of nascent microfibrils) in vivo more effectively, thereby reducing the tissue collagen content to a greater extent.

[0343] Collagen III: collagen I ratio. Although collagen I is the most abundant element of healthy and scar tissues, the fibrils contain other collagen types, most notably collagen III. These collagen types co-assemble into heterotypic fibrils, forming a biological alloy that defines theAttorney Docket No: 205961-7120W01(00553)

[0344] proper architecture and mechanical features of connective tissues. Tn healthy tissues, specific ratios of these collagens vary depending on tissue type and age.

[0345] Numerous studies indicated that these ratios may also change in fibrotic tissues. Due to the non-standardized methods employed by various research groups to assess collagen changes in fibrosis, clear conclusions cannot be drawn regarding the extent or direction of changes in the collagen III to collagen I ratio in the rabbits' PCs in this model. Therefore, collagen III to collagen I ratios were measured in uninjured and injured PCs to assess how different treatments influence this parameter.

[0346] As shown in FIG. 24A, the collagen III to collagen I ratio increased in injured PCs across all treatment groups. However, this increase was markedly smaller in the mACA- and ACA-treated groups compared to the control (FIG. 24B). This minor increase may reflect better preservation of the fibrillar collagen composition in healing PCs from the mACA and ACA groups relative to those from the control group.

[0347] Enumerated Embodiments

[0348] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

[0349] Embodiment 1 provides an isolated polypeptide comprising an antigen-binding domain that specifically binds to the C-terminal telopeptide of the oc2(I) chain of human collagen I, wherein the antigen-binding domain comprises: (a) a heavy chain variable region that comprises three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYTFTDYPLH (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence WINTETGEPTYADD (SEQ ID NO: 2) or WIATETGEPTYADD (SEQ ID NO: 3), and HCDR3 comprises the amino acid sequence GYYYY (SEQ ID NO: 4); and (b) a light chain variable region that comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence KSSQSLLNSRTRKNNL (SEQ ID NO: 4), LCDR2 comprises the amino acid sequence WASTRES (SEQ ID NO: 5), and LCDR3 comprises the amino acid sequence KQSYNLWT (SEQ ID NO: 6).

[0350] Embodiment 2 provides the polypeptide of embodiment 1, wherein the polypeptide comprises (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of theAttorney Docket No: 205961-7120W01(00553)

[0351] heavy chain variable region set forth in SEQ ID NO: 8 or 9; and / or (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 10 or 11.

[0352] Embodiment 3 provides the polypeptide of embodiment 1 or 2, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or 9 and / or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10 or 11.

[0353] Embodiment 4 provides the polypeptide of any one of embodiments 1-3, wherein the polypeptide is a Fab, a single-chain variable fragment (scFv), or an antibody.

[0354] Embodiment 5 provides the polypeptide of any one of embodiments 1-4, comprising human antibody framework regions.

[0355] Embodiment 6 provides the polypeptide of any one of embodiments 1-5, comprising a human antibody constant region.

[0356] Embodiment 7 provides the polypeptide of embodiment 6, wherein the constant region is an IgG constant region.

[0357] Embodiment 8 provides the polypeptide of embodiment 7, wherein the constant region is an IgG4 constant region.

[0358] Embodiment 9 provides the polypeptide of any one of embodiments 1-8, comprising an IgG4 heavy chain constant region and an IgG4 light chain constant region.

[0359] Embodiment 10 provides the polypeptide of embodiment 9, wherein the IgG4 heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 12 and the IgG4 light chain constant region comprises the amino acid sequence of SEQ ID NO: 13.

[0360] Embodiment 11 provides the polypeptide of any one of embodiments 1-10, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or 15.

[0361] Embodiment 12 provides the polypeptide of any one of embodiments 1-10, wherein the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 16 or 17.

[0362] Embodiment 13 provides the polypeptide of embodiment 11 or 12, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 16.Attorney Docket No: 205961-7120W01(00553)

[0363] Embodiment 14 provides the polypeptide of any one of embodiments 1-13, wherein the polypeptide further comprises a collagen binding peptide (CBP) comprising the amino acid sequence of any one of SEQ ID NOs: 18-47.

[0364] Embodiment 15 provides the polypeptide of embodiment 14, wherein the CBP comprises the amino acid sequence of SEQ ID NO: 43.

[0365] Embodiment 16 provides the polypeptide of embodiment 14 or 15, wherein the CBP further comprises a linker on one or both ends of the CBP.

[0366] Embodiment 17 provides the polypeptide of embodiment 16, wherein the linker comprises the amino acid sequence of any one of embodiments 48-59.

[0367] Embodiment 18 provides the polypeptide of embodiment 16 or 17, wherein the polypeptide comprises a CBP-linker comprising the amino acid sequence of any one of SEQ ID NOs: 60-62.

[0368] Embodiment 19 provides the polypeptide of embodiment 14 or 15, wherein the polypeptide comprises 2-5 CBPs.

[0369] Embodiment 20 provides the polypeptide of embodiment 19, wherein the 2-5 CBPs comprise one or more linkers comprising the amino acid sequence of any one of SEQ ID NOs: 48-59.

[0370] Embodiment 21 provides the polypeptide of any one of embodiments 14-20, wherein the polypeptide comprises an IgG4 heavy chain constant region and an IgG4 light chain constant region.

[0371] Embodiment 22 provides the polypeptide of embodiment 21, wherein the IgG4 heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 63 or 64.

[0372] Embodiment 23 provides the polypeptide of embodiment 21 or 22, wherein the IgG4 light chain constant region comprises the amino acid sequence of SEQ ID NO: 65.

[0373] Embodiment 24 provides the polypeptide of any one of embodiments 21-23, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 66-69.

[0374] Embodiment 25 provides the polypeptide of any one of embodiments 21-24, wherein the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 70 or 71.Attorney Docket No: 205961-7120W01(00553)

[0375] Embodiment 26 provides the polypeptide of any one of embodiments 21-25, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or 67 and a light chain comprising the amino acid sequence of SEQ ID NO: 70.

[0376] Embodiment 27 provides the polypeptide of embodiment 4, wherein the polypeptide comprises an scFv comprising the heavy chain variable region and the light chain variable region.

[0377] Embodiment 28 provides the polypeptide of embodiment 27, wherein the scFv comprises a linker between the heavy chain variable region and the light chain variable region, and wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 48-59.

[0378] Embodiment 29 provides the polypeptide of embodiment 27, wherein the scFv comprises a linker between the heavy chain variable region and the light chain variable region, and wherein the linker comprises the amino acid sequence of SEQ ID NO: 50, 58, or 59.

[0379] Embodiment 30 provides the polypeptide of any one of embodiments 27-29, wherein the linker connects the carboxy terminus of the heavy chain variable region to the amino terminus of the light chain variable region.

[0380] Embodiment 31 provides the polypeptide of any one of embodiments 27-29, wherein the linker connects the carboxy terminus of the light chain variable region to the amino terminus of the heavy chain variable region.

[0381] Embodiment 32 provides one or more polynucleotides individually or collectively encoding the polypeptide of any one of embodiments 1-31.

[0382] Embodiment 33 provides one or more expression constructs individually or collectively encoding the one or more polynucleotides of embodiment 32, wherein each of the one or more expression constructs comprises an expression vector.

[0383] Embodiment 34 provides the one or more expression constructs of embodiment 33, wherein each expression construct encodes a signal peptide.

[0384] Embodiment 35 provides the one or more expression constructs of embodiment 33, wherein the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 72-74.

[0385] Embodiment 36 provides the one or more expression constructs of any one of embodiments 33-35, comprising a first expression construct encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 75 or 76, and a second expressionAttorney Docket No: 205961-7120W01(00553)

[0386] construct encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO: 77 or 78.

[0387] Embodiment 37 provides the one or more expression constructs of embodiment 36, wherein the first expression construct encodes a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 76 and the second expression construct encodes a light chain variable region comprising the amino acid sequence of SEQ ID NO: 78.

[0388] Embodiment 38 provides a method of treating a disease or disorder associated with excessive collagen fibril formation in a subject, comprising administering to the subject an effective amount of a polypeptide according to any one of embodiments 1-31.

[0389] Embodiment 39 provides the method according to embodiment 38, wherein the disease or disorder comprises scar formation.

[0390] Embodiment 40 provides the method according to embodiment 38 or 39, wherein the disease or disorder comprises fibrosis.

[0391] Embodiment 41 provides the method according to embodiment 40, wherein the fibrosis comprises pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid formation, myocardial infarction, scleroderma / systemic sclerosis, arthrofibrosis, or adhesive capsulitis.

[0392] Embodiment 42 provides the method according to embodiment 40 or 41, wherein the fibrosis results from a surgical procedure.

[0393] Embodiment 43 provides the method according to embodiment 42, wherein the surgery is abdominal surgery, plastic surgery, glaucoma surgery, or surgery for implantation of a medical implant or device.

[0394] Other Embodiments

[0395] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents. The recitation ofAttorney Docket No: 205961-7120W01(00553)

[0396] a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0397] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.Attorney Docket No: 205961-7120W01(00553)

[0398] REFERENCES

[0399] 1. Nesterenko S, Morrey ME, Abdel MP, An KN, Steinmann SP, Morrey BF, Sanchez-Sotelo J.

[0400] New rabbit knee model of posttraumatic joint contracture: indirect capsular damage induces a severe contracture. J Orthop Res. 2009;27(8): 1028-32. PubMed PMID: 19165743.

[0401] 2. Hildebrand KA, Holmberg M, Shrive N. A new method to measure post-traumatic joint contractures in the rabbit knee. Journal of biomechanical engineering. 2003;125(6):887-92. PubMed PMID: 14986415.

[0402] 3. Monument MJ, Hart DA, Befus AD, Salo PT, Zhang M, Hildebrand KA. The mast cell stabilizer ketotifen reduces joint capsule fibrosis in a rabbit model of post-traumatic joint contractures. Inflamm Res. 2012;61(4):285-92. PubMed PMID: 22173279; PubMed Central PMCID: PMC3710138.

[0403] 4. Hildebrand KA, Zhang M, Hart DA. Joint capsule matrix turnover in a rabbit model of chronic joint contractures: Correlation with human contractures. J Orthop Res.

[0404] 2006;24(5): 1036-43. PubMed PMID: 16596651; PubMed Central PMCID: PMC2950172. 5. Steplewski A, Fertala J, Cheng L, Wang ML, Rivlin M, Beredjiklian P, Fertala A. Evaluating the Efficacy of a Thermoresponsive Hydrogel for Delivering Anti-Collagen Antibodies to Reduce Posttraumatic Scarring in Orthopedic Tissues. Gels. 2023;9(12):97L PubMed PMID: 38131957; PubMed Central PMCID: PMC 10742524

[0405] 6. Steplewski A, Fertala J, Tomlinson RE, Wang ML, Donahue A, Arnold WV, Rivlin M, Beredjiklian PK, Abboud JA, Namdari S, Fertala A. Mechanisms of reducing joint stiffness by blocking collagen fibrillogenesis in a rabbit model of posttraumatic arthrofibrosis. PLoS One. 2021;16(9):e0257147. PubMed PMID: 34492074; PubMed Central PMCID:

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[0407] 7. Steplewski A, Fertala J, Beredjiklian PK, Abboud JA, Wang ML, Namdari S, Barlow J, Rivlin M, Arnold WV, Kostas J, Hou C, Fertala A. Auxiliary proteins that facilitate formation of collagen-rich deposits in the posterior knee capsule in a rabbit-based joint contracture model. J Orthop Res. 2016;34(3):489-501. PubMed PMID: 26241613.

[0408] 8. Steplewski A, Fertala J, Beredjiklian PK, Abboud JA, Wang MLY, Namdari S, Barlow J, Rivlin M, Arnold WV, Kostas J, Hou C, Fertala A. Blocking collagen fibril formation inAttorney Docket No: 205961-7120W01(00553)

[0409] injured knees reduces flexion contracture in a rabbit model. J Orthop Res. 2017;35(5): 1038- 46. PubMed PMID: 27419365.

[0410] 9. Hildebrand KA, Zhang M, Germscheid NM, Wang C, Hart DA. Cellular, matrix, and growth factor components of the joint capsule are modified early in the process of posttraumatic contracture formation in a rabbit model. Acta orthopaedica. 2008;79(l): 116-25. PubMed PMID: 18283583; PubMed Central PMCID: PMC2950862.

[0411] 10. Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. The Histochemical journal.

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[0413] 11. Jalil JE, Doering CW, Janicki JS, Pick R, Shroff SG, Weber KT. Fibrillar collagen and myocardial stiffness in the intact hypertrophied rat left ventricle. Circ Res. 1989;64(6): 1041- 50. PubMed PMID: 2524288.

[0414] 12. Gopinathan PA, Kokila G, Jyothi M, Ananjan C, Pradeep L, Nazir SH. Study of collagen birefringence in different grades of oral squamous cell carcinoma using picrosirius red and polarized light microscopy. Scientifica. 2015;2015:802980-. PubMed PMID.

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[0417] 15. Montes GS, Nicolelis MA, Brentani-Samaia HP, Furuie SS. Collagen fibril diameters in arteries of mice. A comparison of manual and computer-aided morphometric analyses. Acta Anat (Basel). 1989; 135(1):57-61. PubMed PMID: 2750461.

[0418] 16. Allon I, Vered M, Buchner A, Dayan D. Stromal differences in salivary gland tumors of a common histopathogenesis but with different biological behavior: a study with picrosirius red and polarizing microscopy. Acta Histochem. 2006;108(4):259-64. PubMed PMID:

[0419] 16899283.Attorney Docket No: 205961-7120W01(00553)

[0420] 17. Rich L, Whittaker P. Collagen and picrosirius red staining: A polarized light assessment of fibrillar hue and spatial distribution. Journal of Morphological Sciences. 2005;22(2):97-104. PubMed PMID.

[0421] 18. Querido W, Kandel S, Pleshko N. Applications of Vibrational Spectroscopy for Analysis of Connective Tissues. Molecules. 2021;26(4). PubMed PMID: 33572384; PubMed Central PMCID: PMCPMC7916244.

[0422] 19. Cheheltani R, Rosano JM, Wang B, Sabri AK, Pleshko N, Kiani MF. Fourier transform infrared spectroscopic imaging of cardiac tissue to detect collagen deposition after myocardial infarction. J Biomed Opt. 2012;17(5):056014. PubMed PMID: 22612137;

[0423] PubMed Central PMCID: PMC3381018.

[0424] 20. Steplewski A, Fertala J, Tomlinson R, Hoxha K, Han L, Thakar O, Klein J, Abboud J, Fertala A. The impact of cholesterol deposits on the fibrillar architecture of the Achilles tendon in a rabbit model of hypercholesterolemia. Journal of orthopaedic surgery and research.

[0425] 2019;14(l): 172. PubMed PMID: 31182124; PubMed Central PMCID: PMC6558834.

[0426] 21. Paschalis EP, Verdelis K, Doty SB, Boskey AL, Mendelsohn R, Yamauchi M. Spectroscopic characterization of collagen crossdinks in bone. J Bone Miner Res. 2001; 16(10): 1821-8. PubMed PMID: 11585346.

[0427] 22. Eyre DR, Paz MA, Gallop PM. Crossdinking in collagen and elastin. Annu Rev Biochem.

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[0429] 23. van der Slot AJ, van Dura EA, de Wit EC, De Groot J, Huizinga TW, Bank RA, Zuurmond AM. Elevated formation of pyridinoline cross-links by profibrotic cytokines is associated with enhanced lysyl hydroxylase 2b levels. Biochim Biophys Acta. 2005;1741(l-2):95-102. PubMed PMID: 15955452.

[0430] 24. van der Slot AJ, Zuurmond AM, van den Bogaerdt AJ, Ulrich MM, Middelkoop E, Boers W, Karel Ronday H, DeGroot J, Huizinga TW, Bank RA. Increased formation of pyridinoline cross-links due to higher telopeptide lysyl hydroxylase levels is a general fibrotic phenomenon. Matrix Biol. 2004;23(4):251-7. PubMed PMID: 15296939.Attorney Docket No: 205961-7120W01(00553)

[0431] 25. Brickley -Parsons D, Glimcher MJ, Smith RJ, Albin R, Adams JP. Biochemical changes in the collagen of the palmar fascia in patients with Dupuytren's disease. J Bone Joint Surg Am.

[0432] 1981;63(5):787-97. PubMed PMID: 7240301.

[0433] 26. Rieppo L, Saarakkala S, Narhi T, Helminen HJ, Jurvelin JS, Rieppo J. Application of second derivative spectroscopy for increasing molecular specificity of Fourier transform infrared spectroscopic imaging of articular cartilage. Osteoarthritis Cartilage. 2012;20(5):451-9.

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[0435] 27. Gieroba B, Przekora A, Kalisz G, Kazimierczak P, Song CL, Wojcik M, Ginalska G, Kazarian SG, Sroka-Bartnicka A. Collagen maturity and mineralization in mesenchymal stem cells cultured on the hydroxyapatite-based bone scaffold analyzed by ATR-FTIR spectroscopic imaging. Mater Sci Eng C Mater Biol Appl. 2021 ; 119:111634. PubMed PMID: 33321672.

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[0438] 30. Viele K, Berry S, Neuenschwander B, Amzal B, Chen F, Enas N, Hobbs B, Ibrahim JG, Kinnersley N, Lindborg S. Use of historical control data for assessing treatment effects in clinical trials. Pharmaceutical statistics. 2014; 13(l):41-54. PubMed PMID.

[0439] 31. Chung HJ, Steplewski A, Chung KY, Uitto J, Fertala A. Collagen fibril formation. A new target to limit fibrosis. J Biol Chem. 2008;283(38):25879-86. PubMed PMID: 18650436; PubMed Central PMCID: PMC2533774.

[0440] 32. Frank C, McDonald D, Shrive N. Collagen fibril diameters in the rabbit medial collateral ligament scar: a longer term assessment. Connect Tissue Res. 1997;36(3):261 -9. PubMed PMID: 9512894.Attorney Docket No: 205961-7120W01(00553)

[0441] Puente A, Fortea JI, Cabezas J, Arias Loste MT, Iruzubieta P, Llerena S, Huelin P, Fabrega E, Crespo J. L0XL2-A New Target in Antifibrogenic Therapy? Int J Mol Sci. 2019;20(7). PubMed PMID: 30986934; PubMed Central PMCID: PMCPMC6480111.

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Claims

Attorney Docket No: 205961-7120W01(00553)CLAIMSWhat is claimed:

1. An isolated polypeptide comprising an antigen-binding domain that specifically binds to the C-terminal telopeptide of the oc2(I) chain of human collagen I, wherein the antigen-binding domain comprises:(a) a heavy chain variable region that comprises three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYTFTDYPLH (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence WINTETGEPTYADD (SEQ ID NO: 2) or WIATETGEPTYADD (SEQ ID NO: 3), and HCDR3 comprises the amino acid sequence GYYYY (SEQ ID NO: 4); and(b) a light chain variable region that comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence KSSQSLLNSRTRKNNL (SEQ ID NO: 4), LCDR2 comprises the amino acid sequence WASTRES (SEQ ID NO: 5), and LCDR3 comprises the amino acid sequence KQSYNLWT (SEQ ID NO: 6).

2. The polypeptide of claim 1, wherein the polypeptide comprises (a) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 8 or 9; and / or (b) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 10 or 11.

3. The polypeptide of claim 1 or 2, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or 9 and / or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10 or 11.

4. The polypeptide of any one of claims 1-3, wherein the polypeptide is a Fab, a singlechain variable fragment (scFv), or an antibody.Attorney Docket No: 205961-7120W01(00553)5. The polypeptide of any one of claims 1-4, comprising human antibody framework regions.

6. The polypeptide of any one of claims 1-5, comprising a human antibody constant region.

7. The polypeptide of claim 6, wherein the constant region is an IgG constant region.

8. The polypeptide of claim 7, wherein the constant region is an IgG4 constant region.

9. The polypeptide of any one of claims 1-8, comprising an IgG4 heavy chain constant region and an IgG4 light chain constant region.

10. The polypeptide of claim 9, wherein the lgG4 heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 12 and the IgG4 light chain constant region comprises the amino acid sequence of SEQ ID NO: 13.

11. The polypeptide of any one of claims 1-10, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or 15.

12. The polypeptide of any one of claims 1-10, wherein the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 16 or 17.

13. The polypeptide of claim 11 or 12, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 16.

14. The polypeptide of any one of claims 1-13, wherein the polypeptide further comprises a collagen binding peptide (CBP) comprising the amino acid sequence of any one of SEQ ID NOs: 18-47.

15. The polypeptide of claim 14, wherein the CBP comprises the amino acid sequence of SEQ ID NO: 43.Attorney Docket No: 205961-7120W01(00553)16. The polypeptide of claim 14 or 15, wherein the CBP further comprises a linker on one or both ends of the CBP.

17. The polypeptide of claim 16, wherein the linker comprises the amino acid sequence of any one of claims 48-59.

18. The polypeptide of claim 16 or 17, wherein the polypeptide comprises a CBP-linker comprising the amino acid sequence of any one of SEQ ID NOs: 60-62.

19. The polypeptide of claim 14 or 15, wherein the polypeptide comprises 2-5 CBPs.

20. The polypeptide of claim 19, wherein the 2-5 CBPs comprise one or more linkers comprising the amino acid sequence of any one of SEQ ID NOs: 48-59.

21. The polypeptide of any one of claims 14-20, wherein the polypeptide comprises an IgG4 heavy chain constant region and an IgG4 light chain constant region.

22. The polypeptide of claim 21, wherein the IgG4 heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 63 or 64.

23. The polypeptide of claim 21 or 22, wherein the IgG4 light chain constant region comprises the amino acid sequence of SEQ ID NO: 65.

24. The polypeptide of any one of claims 21-23, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 66-69.

25. The polypeptide of any one of claims 21-24, wherein the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 70 or 71.

26. The polypeptide of any one of claims 21-25, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or 67 and a light chain comprising the amino acid sequence of SEQ ID NO: 70.Attorney Docket No: 205961-7120W01(00553)27. The polypeptide of claim 4, wherein the polypeptide comprises an scFv comprising the heavy chain variable region and the light chain variable region.

28. The polypeptide of claim 27, wherein the scFv comprises a linker between the heavy chain variable region and the light chain variable region, and wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 48-59.

29. The polypeptide of claim 27, wherein the scFv comprises a linker between the heavy chain variable region and the light chain variable region, and wherein the linker comprises the amino acid sequence of SEQ ID NO: 50, 58, or 59.

30. The polypeptide of any one of claims 27-29, wherein the linker connects the carboxy terminus of the heavy chain variable region to the amino terminus of the light chain variable region.

31. The polypeptide of any one of claims 27-29, wherein the linker connects the carboxy terminus of the light chain variable region to the amino terminus of the heavy chain variable region.

32. One or more polynucleotides individually or collectively encoding the polypeptide of any one of claims 1-31.

33. One or more expression constructs individually or collectively encoding the one or more polynucleotides of claim 32, wherein each of the one or more expression constructs comprises an expression vector.

34. The one or more expression constructs of claim 33, wherein each expression construct encodes a signal peptide.

35. The one or more expression constructs of claim 33, wherein the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 72-74.Attorney Docket No: 205961-7120W01(00553)36. The one or more expression constructs of any one of claims 33-35, comprising a first expression construct encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 75 or 76, and a second expression construct encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO: 77 or 78.

37. The one or more expression constructs of claim 36, wherein the first expression construct encodes a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 76 and the second expression construct encodes a light chain variable region comprising the amino acid sequence of SEQ ID NO: 78.

38. A method of treating a disease or disorder associated with excessive collagen fibril formation in a subject, comprising administering to the subject an effective amount of a polypeptide according to any one of claims 1-31.

39. The method according to claim 38, wherein the disease or disorder comprises scar formation.

40. The method according to claim 38 or 39, wherein the disease or disorder comprises fibrosis.

41. The method according to claim 40, wherein the fibrosis comprises pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid formation, myocardial infarction, scleroderma / systemic sclerosis, arthrofibrosis, or adhesive capsulitis.

42. The method according to claim 40 or 41, wherein the fibrosis results from a surgical procedure.

43. The method according to claim 42, wherein the surgery is abdominal surgery, plastic surgery, glaucoma surgery, or surgery for implantation of a medical implant or device.

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