Treatment of moderate to severe osteogenesis imperfecta

Abstract

The present disclosure provides methods for treating and ameliorating moderate to severe osteogenesis imperfecta (OI) in a subject by administering to the subject a therapeutically effective amount of an agent that binds and neutralizes transforming growth factor beta (TGFβ).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 185,967, filed May 7, 2021. The disclosure of this priority application is incorporated herein by reference in its entirety.

[0002] Government funding This invention was made with government support under Grant AR068069 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

[0003] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated by reference in its entirety. Said ASCII copy, created on May 3, 2022, is named 022548_WO026_SL.txt and is 17,875 bytes in size.

[0004] joint research agreement This work was supported by a research agreement with Sanofi Genzyme. [Background technology]

[0005] Osteogenesis imperfecta (OI) is a genetically and phenotypically heterogeneous Mendelian disease of fusion disorders with an estimated prevalence of 1 in 10,000-20,000 births. Skeletal symptoms of OI include low bone mass, bone fragility, recurrent fractures, scoliosis, and bone deformities. Extraskeletal symptoms include low muscle mass, muscle weakness, odontogenesis imperfecta, hearing loss, and pulmonary disease (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8). Management of individuals with OI usually involves a multidisciplinary approach. The backbone therapy for OI bone fragility involves repurposing medications used to treat osteoporosis (Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11; Non-Patent Document 12; Non-Patent Document 13; Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 17).

[0006] Bisphosphonates (BPN), a class of antiresorptive drugs that reduce bone remodeling, have become the standard of care, especially in pediatric OI. In children, BPN has been shown to have beneficial effects on areal and volumetric bone mineral density (aBMD and vBMD), progression of scoliosis, quality of life, and in some studies, fracture incidence (Non-Patent Document 18; Non-Patent Document 14; Non-Patent Document 19; Non-Patent Document 20). However, given the heterogeneity of OI and the variability of clinical trial designs, the effects of BPN are inconsistent. In adults, the benefits and outcomes of long-term treatment with bisphosphonates are less certain (Non-Patent Document 9; Non-Patent Document 21). In addition, in randomized trials involving adults with OI, treatment with the anabolic agent teriparatide led to improvements in aBMD and vBMD in individuals with mild (OI type I) but not in those with moderate to severe forms of the disorder (OI types III and IV). Furthermore, none of these repurposed therapies address the specific pathogenic mechanisms in OI and therefore have any effect on the extraskeletal manifestations. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Marini, Nat Rev Dis Primers (2017)3:17052 p. [Non-Patent Document 2] Marom et al., Am J Med Genet C Semin Med Genet. (2016) 172(4):367-83 [Non-Patent Document 3] Patel et al., Clin Gen. (2015) 87(2):133-40 [Non-Patent Document 4] Rossi et al., Curr Opin Pediatr. (2019) 31(6):708~15 [Non-Patent Document 5] Tam et al., Clin Gen. (2018) 94(6):502-11 [Non-Patent Document 6] DiMeglio et al. J Bone Miner Res. (2006) 21:132-40 [Non-Patent Document 7] Gatti et al., J Bone Miner Res. (2005) 20(5):758-63 [Non-Patent Document 8] Gatti et al., Calcified Tissue Int. (2013) 93(5):448-52 [Non-Patent Document 9] Adami et al., J Bone Miner Res. (2003) 18(1):126-30 [Non-Patent Document 10] Bishop et al., Ear Hum Dev. (2010) 86(11):743-6 [Non-Patent Document 11] Chevrel et al., J Bone Miner Res. (2006) 21(2): pp. 300-6. [Non-Patent Document 12] Glorieux et al., NEJM (1998) 339(14):947-52 [Non-Patent Document 13] Rauch et al., J Bone Miner Res. (2009) 24(7):1282-9 [Non-Patent Document 14] Rauch et al., J Bone Miner Res. (2003) 18(4):610-4 [Non-Patent Document 15] Orwoll et al., J Clin Invest (2014) 124(2):491-8 [Non-Patent Document 16] Hoyer-Kuhn et al., J Musculoskelet Neuronal Interact / (2016)16(1):24-32 [Non-Patent Document 17] Anissipour et al., J Bone Joint Surg Am. (2014) 96(3):237-43 [Non-Patent Document 18] Bishop et al., Lancet (2013) 382(9902):1424-32 [Non-Patent Document 19] Bains et al., JBMR Plus(2019)3(5):e10118 [Non-Patent Document 20] Rauch et al., Bone (2007) 40(2):274-80 [Non-Patent Document 21] Shi et al., Am J Ther. (2016) 23(3):e894-904 Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there remains a serious unmet need for effective therapies targeting moderate to severe OI. [Means for solving the problem]

[0009] The present disclosure provides a method for treating osteogenesis imperfecta (OI) in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-TGFβ antibody or antigen-binding fragment thereof, wherein the anti-TGFβ antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (CDRs) 1 to 3 comprising SEQ ID NOs: 4 to 6, respectively, and light chain CDRs 1 to 3 comprising SEQ ID NOs: 7 to 9, respectively, and wherein the therapeutically effective amount is 1 to 10 mg / kg.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable domain comprising SEQ ID NO: 10 and a light chain variable domain comprising SEQ ID NO: 11. In some embodiments, the antibody comprises a human IgG4 constant region and / or a human kappa light chain constant region. In certain embodiments, the human IgG4 constant region comprises a S228P mutation (Eu numbering). In certain embodiments, the antibody comprises a heavy chain comprising SEQ ID NO: 1 and a light chain comprising SEQ ID NO: 2. In other certain embodiments, the antibody comprises a heavy chain comprising SEQ ID NO: 3 and a light chain comprising SEQ ID NO: 2.

[0011] In some embodiments, the antibody comprises a bone targeting moiety, and in some cases the bone targeting moiety is a polyarginine peptide.In some embodiments, the antibody comprises one or more polyarginine peptides, for example, at the N-terminus, or C-terminus, or both termini of the heavy chain, and / or at the C-terminus of the light chain, of the antibody or antigen-binding fragment.In certain embodiments, the polyarginine peptide is D10 (SEQ ID NO: 14).

[0012] In some embodiments, the OI is moderate to severe OI or type IV OI. In some embodiments, the human subject is an adult patient (≧18 years old) or a pediatric patient (<18 years old). In some embodiments, the human subject has a mutation in the COL1A1 or COL1A2 gene, and optionally, the mutation is a glycine substitution mutation in the COL1A1 or COL1A2 gene, or a valine deletion in the COL1A2 gene.

[0013] In some embodiments, administration improves a bone parameter selected from the group consisting of bone mineral density (BMD), bone mass density (BV / TV), total bone surface (BS), bone surface density (BS / BV), trabecular number (Tb.N), trabecular width (Tb.Th), trabecular center distance (Tb.Sp), and total bone volume (Dens TV). In further embodiments, the bone parameter is lumbar spine area BMD (LS aBMD), and optionally LS aBMD is improved by at least 1-10% after administration compared to baseline levels.

[0014] In some embodiments, the administration reduces bone turnover and / or bone cell density, optionally where reduced bone turnover is indicated by a decrease in serum CTX or an increase in serum osteocalcin (OCN).

[0015] In some embodiments, the administering step is repeated monthly, every two months, every three months, every six months, every nine months, or every twelve months, e.g., at a dose of 4 mg / kg. The antibody or antigen-binding fragment can be administered by intravenous infusion.

[0016] In some embodiments, the method further includes administering to the subject another therapeutic agent, such as a bisphosphonate (e.g., alendronate, pamidronate, zoledronate, and risedronate), parathyroid hormone, calcitonin, teriparatide, or an anti-sclerostin agent.

[0017] Also provided herein are anti-TGFβ antibodies, or antigen-binding fragments thereof, for use in the treatment of osteogenesis imperfecta in the methods of treatment herein, and the use of anti-TGFβ antibodies, or antigen-binding fragments thereof, in the manufacture of a medicament for treating osteogenesis imperfecta in the methods of treatment herein.

[0018] Also provided are articles of manufacture (eg, kits) that include an anti-TGFβ antibody, or antigen-binding fragment thereof, for use in the treatment methods herein for treating osteogenesis imperfecta.

[0019] Other features, objects and advantages of the present invention will be apparent in the following detailed description of the invention. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of illustration only and is not limiting. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0020] [Figure 1] Figure 1. Human bone specimen processing for histology, RNA, and protein studies. Panel A: An illustration of the bone specimen milling environment showing the bone specimen immersed in two layers of liquid nitrogen and milled with a power drill. Panel B: An image of the bone specimen before processing is shown in the upper right corner. After milling, the bone powder at the bottom of the pestle was collected in liquid nitrogen. A 2-3 mm3 area immediately adjacent to the milled area was processed for histology and immunochemistry. [Figure 2-1] Bone histology of OI type III individuals and non-diseased controls. Panel A: Full images of H&E stained tissue sections of harvested bone specimens. The majority of specimens from non-diseased individuals were cortical bone. OI type III specimens were more heterogeneous. Most were cortical bone, but two specimens (OI62 and OI83) contained only trabecular bone. One specimen (OI41) had fibrous cartilage and one specimen (OI35) contained callus and trabecular bone. Scale bar: 200 μm. Panel B: Representative higher magnification (10×) H&E stained images of non-diseased control bone and OI type III bone. In the control bone, a well-organized Haversian canal system was observed, while OI type III bone showed a more fibrous cortical bone with less organized Haversian canals and more osteocytes that were more spherical in shape. Scale bar: 50 μm. [Figure 2-2] Continued from Figure 2-1. [Diagram 3] FIG. 1 shows increased osteocyte density in type III OI bone. Quantification of osteocyte density in unaffected controls (n=4) and type III OI (n=10). Each dot represents the osteocyte density of one individual. Means and standard deviations are shown. [Figure 4-1] Figure 1. Expression changes of genes using Nanostring and RNA Sequence platforms. One non-OI control and one type III OI were included in the analysis along with both RNA-seq and NanoString data. 155 genes that met NanoString quality control were used for validation of RNA-seq differential expression data. Direction of fold change (up or down compared to non-OI control) from RNA-seq and NanoString for 155 genes is presented as red up arrow (up in type III OI) or blue down arrow (down in type III OI). Purple background: direction of fold change discordant between the two platforms. White background: direction of fold change concordant between the two platforms. Concordance is 92%. [Figure 4-2] Continued from Figure 4-1. [Figure 4-3] Continued from Figure 4-2. [Figure 4-4] Continued from Figure 4-3. [Figure 4-5] Continued from Figure 4-4. [Figure 5-1] Figure 1 depicts transcriptomic and bioinformatics analyses demonstrating activation of TGFβ signaling in OI type III bones. Panel A shows principal component analysis (PCA) plots of all controls and OI type III in 3-PC dimensions. Panel B shows hierarchical clustering based on Euclidean distance using RPKM of all control and OI type III bone data. Blue: downregulated; Yellow: upregulated. Panel C shows gene set enrichment plots demonstrating activation of TGFβ signaling. NES: normalized enrichment score. FDR: false discovery rate. Expression pattern of genes involved in TGFβ genes set in the analysis database. Blue: downregulated. Red: upregulated. C: Control. OI: OI type III. [Figure 5-2] Continued from Figure 5-1. [Figure 5-3] Continued from Figure 5-2. [Figure 6-1]Figure 1 shows significantly enriched Gene Ontology (GO) analysis results with enrichment fold change in type III OI bone. Partek GO enrichment results based on skeletal process, osteocyte, collagen, and skeletal-related major signaling pathway categories are provided. Significance was defined by a P value <0.05. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 6-4] Continued from Figure 6-3. [Figure 6-5] Continued from Figure 6-4. [Figure 7-1] Figure 2 shows the top 20 Gene Set Enrichment Assay (GSEA) results of transcriptional profiles of type III OI and controls. The top 20 enriched gene sets in type III OI or in non-OI controls are provided. NES: normalized enrichment score. FDR q-val: false discovery rate adjusted q-value. [Figure 7-2] Continued from Figure 7-1. [Figure 8-1] Figure 1 shows significantly changed proteins in RPPA analysis. The table shows the complete list of significantly changed proteins based on a nominal P value of <0.05 in type III OI bone. Protein expression levels are presented as normalized intensity of protein arrays. [Figure 8-2] Continued from Figure 8-1. [Figure 9]Figure 1. Elevated levels of phosphorylated SMAD2 (pSMAD2) in type III bone from OI. Panel A shows immunohistochemical staining of pSMAD2 in control and OI type III bone sections. Higher magnification images are shown in the black box at the bottom right. Elevated pSMAD2 signal was detected in all OI samples, especially in osteocytes. Scale bar: 20 μm. Panel B shows Western blots of phosphorylated SMAD2 (p-SMAD2) and total SMAD2 (T-SMAD2) in protein extracted from control and OI type III bone. 50 μg of total protein was loaded. An additional OI62 sample was treated with calf intestinal alkaline phosphatase (CIP) to remove the phosphorylated signal and served as a negative control for accurate pSMAD2 signal (indicated by arrow). Panel C shows quantification of the Western blot from (B) but shown as a ratio of phosphorylated (phospho) to total SMAD2. GAPDH was used as a loading control. C: Control. OI: OI type III. [Figure 10] Figure 1 shows hematological safety data from a study evaluating the safety of fresolimumab in adults with OI. Hemoglobin drop was observed in two participants (FR005 and FR009), both of whom were classified as mild. These two individuals had epistaxis that was classified as drug-related and menstrual bleeding that was not classified as drug-related. Platelet counts and INR were within normal ranges. [Figure 11] Figure 3 illustrates the effect of fresolimumab on bone turnover markers and bone mineral density. Increases in osteocalcin (Ocn) and C-terminal telopeptide (CTX) levels were observed in three of four participants in the 1 mg·1 kg bw-1 cohort, with peak values ​​observed between 30 and 90 days after treatment. In the 4 mg·1 kg bw-1 cohort, a decrease in Ocn was observed at day 30, and this suppression persisted through day 180. In participant FR012, it was not possible to obtain bone turnover markers at day 30 due to the inability to ambulate. Two individuals with type IV OI had robust improvements in L1-4 aBMD in both dose cohorts. [Figure 12]FIG. 1 illustrates the correlation and agreement between the two median readings of LS aBMD. LS aBMD was read by two independent readers blinded to the study design. There was a strong correlation between the two readings (R=0.995). Bland-Altman plots showed a high degree of agreement between the two readings. The mean difference, upper limit of agreement, lower limit of agreement, and confidence intervals are illustrated in blue, green, and red, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present disclosure provides a method of treating moderate to severe OI (e.g., type IV OI) in a human patient by administering a monoclonal antibody that binds to and neutralizes all isoforms of human TGFβ. The method is based on the surprising discovery that infrequent dosing of an anti-TGFβ antibody (e.g., every 3 or 6 months) can be sufficient to ameliorate symptoms of OI in a patient.

[0022] Standard treatment therapies for OI bone fragility include repurposing medications used to treat osteoporosis. However, given the heterogeneity of OI and the variability of clinical trial design, the efficacy of BPN is inconsistent. Furthermore, in a randomized study involving adults with OI, treatment with the anabolic agent teriparatide resulted in improved aBMD and vBMD in individuals with milder disease (OI type I) but not in those with moderate to severe disease (OI type III and type IV). None of these repurposed therapies address the specific pathogenic mechanisms in OI and therefore have no effect on extraskeletal symptoms. The inventors have surprisingly found that individuals with moderate to severe OI treated with a single dose of 1 or 4 mg / kg anti-TFGp antibody showed robust improvement in lumbar spine areal bone mineral density (LS aBMD).

[0023] Targeting TGFβ signaling in bone offers important pharmacodynamic advantages. Sustained pharmacological inhibition is necessary to modulate such important pathways in extraskeletal tissues, but the remodeling unit of human bone is approximately 3 months. Thus, pharmacological inhibition at a single time point may have long-term effects beyond the terminal half-life and persistence of the drug in circulation. Herein, it is shown that treatment with a single dose of a pan-specific anti-TGFβ antibody was associated with changes in bone turnover and aBMD at 90 and 180 days. In addition, less frequent dosing provides safety benefits due to lower cumulative dosages, which in turn allows for reduced systemic toxicity. The efficacy of less frequent dosing is surprising, because in preclinical studies in OI mice, treatment with an anti-TGFβ antibody (mouse antibody 1D11) at a frequency of 3 times a week provided findings of improvement, but the improvement was attenuated when the mice were dosed less frequently (e.g., Q4W).

[0024] I. Osteogenesis imperfecta OI encompasses a group of congenital bone disorders characterized by the deficiency of one or more proteins involved in bone matrix deposition or homeostasis. There are over 19 types of OI defined by the specific gene mutation, the deficiency of the protein produced, and the phenotype of the affected individual. Classification includes findings on x-rays and other imaging studies. The main types of OI are as follows (information from the John Hopkins University website):

[0025] Type I is the mildest and most common type. Approximately 50% of all affected children have this type. Fractures and deformities are rare.

[0026] Type II is the most severe type. Infants have very short arms and legs, a small chest, and a soft skull. Infants may be born with fractures, have low birth weight, and underdeveloped lungs. Infants with Type II OI usually die within the first few weeks of life.

[0027] Type III is the most severe type in infants who do not die during the neonatal period. At birth, infants may have arms that are slightly shorter than normal and fractures to the arms, legs, and ribs. Infants may also have a larger than normal head, a triangular-shaped face, a deformed chest and spine, and problems breathing and swallowing.

[0028] Type IV is a type of OI where symptoms range from mild to severe. Babies with Type IV may be diagnosed at birth. Babies may not break any bones until they begin to crawl or walk. The bones in the arms and legs may not be straight. Babies may not grow normally.

[0029] Type V is similar to Type IV. Symptoms can be moderate to severe. It is common to have areas of thickening (callus hypertrophy) in the area where the large bones are fractured.

[0030] Type VI is very rare. Symptoms are mild and similar to type IV.

[0031] Type VII can be similar to Type IV or Type II. It is common for people to have shorter than normal height. It is also common for people to have shorter than normal arms and femurs.

[0032] Type VIII is similar to types II and III. Patients have very soft bones and severe growth problems.

[0033] Although the phenotype varies among types of OI, common symptoms include incomplete ossification of the skeleton and teeth, bone loss, weak bones, and pathological fractures. Specific symptoms include bones that break easily, bone deformities (e.g., bowed legs), discoloration of the whites of the eyes (sclera), barrel chest, curved spine, triangular-shaped face, loose joints, muscle weakness, skin that bruises easily, hearing loss in early adulthood, and / or soft, discolored teeth. Complications of OI include respiratory infections (e.g., pneumonia), heart problems (e.g., poor heart valve function), kidney stones, joint problems, hearing loss, and abnormal eye conditions (including blindness). OI can be diagnosed or monitored by x-rays, laboratory tests (e.g., blood and genetic tests), dual-energy x-ray absorptiometry scans (DXA or DEXA scans), and bone biopsies.

[0034] Although multiple pathogenic gene mutations can cause various subtypes of OI, >90% are caused by pathogenic variants in the COL1A1 gene (encoding type I collagen alpha 1 chain) or COL2A1 gene (encoding type II collagen alpha 1 chain), or in genes encoding proteins that post-translationally modify type I collagen (CRTAP, PPIB, and LEPRE1) (Patel et al., ibid.; Lim et al., Bone (2017) 102:40-49).

[0035] The treatment method of the present disclosure is effective in treating moderate to severe OI, such as type IV OI.In some embodiments, the OI of the patient is caused by mutation (e.g., glycine substitution) in COL1A1 or COL1A2, or by biallelic pathogenic variants in CRTAP, PPIB or LEPRE1.See, for example, the mutations shown in Tables 1 and 3 below.

[0036] II. Anti-TGFβ antibody TGFβ is a multifunctional cytokine involved in cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses. Secreted TGFβ protein is cleaved into latency-associated peptide (LAP) and mature TGFβ peptide, which is found in latent and active forms. Mature TGFβ peptide forms both homodimers and heterodimers with other TGFβ family members.

[0037] There are three human (h)TGFβ isoforms: TGFβ1, TGFβ2 and TGFβ3 (UniProt accession numbers P01137, P08112 and P10600, respectively). TGFβ1 differs from TGFβ2 by 27 and from TGFβ3 by 22, mostly conservative amino acids. Human TGFβ is highly similar to mouse TGFβ: human TGFβ1 has only one amino acid difference from mouse TGFβ1; human TGFβ2 has only three amino acid differences from mouse TGFβ2; and human TGFβ3 is identical to mouse TGFβ3.

[0038] Binding of TGFβ proteins to homodimeric or heterodimeric TGFβ transmembrane receptor complexes activates the canonical TGFβ signaling pathway mediated by intracellular SMAD proteins. Deregulation of TGFβ leads to pathological processes in humans that have been implicated in a variety of conditions, such as birth defects, cancer, chronic inflammation, autoimmune diseases, and fibrotic diseases (see, e.g., Border et al., Curr Opin Nephrol Hypertens. (1994) 3(4):446-52; Border et al., Kidney Int Supplement (1995) 49:S59-61).

[0039] For the present OI treatment method, the anti-TGFβ antibody can be a pan-specific antibody, i.e., an antibody that binds with high affinity to and neutralizes all three isoforms of TGFβ. In some embodiments, the antibody is fresolimumab. Fresolimumab is a recombinant human antibody. Its heavy chain is shown below:

[0040] [ka] In the above sequence, positions 1-120 correspond to the heavy chain variable domain (V H ), with the heavy chain CDRs ("HCDRs"; Kabat definition) boxed. The heavy chain comprises a human IgG4 constant region.

[0041] The light chain of fresolimumab is shown below:

[0042] [ka] In the above sequence, positions 1-108 correspond to the light chain variable domain (V L ), with the light chain CDRs ("LCDRs"; Kabat definition) underlined. The light chain comprises a human Cκ constant region.

[0043] In some embodiments, the anti-TGFβ antibody herein is a variant of fresolimumab, Ab1. The heavy chain of Ab1 differs from that of fresolimumab only in one residue in the IgG4 hinge region. The residue is S228 (Eu numbering), where Ab1 has a proline at that position, i.e., an S228P substitution compared to fresolimumab. Ab1 and fresolimumab have the same light chain. The heavy chain of Ab1 is shown below:

[0044] [ka] In the above sequence, the HCDRs are boxed and the S228P substitution is boxed and in bold.

[0045] In some embodiments, the anti-TGFβ antibody comprises one or more (e.g., all six) of HCDRs 1-3 and LCDRs 1-3 of fresolimumab. In other words, the antibody comprises one or more (e.g., all six) of the following HCDRs and LCDRs: HCDR1 SNVIS (SEQ ID NO: 4) HCDR2 GVIPIVDIANYAQRFKG (SEQ ID NO: 5) HCDR3 TLGLVLDAMDY (SEQ ID NO: 6) LCDR1 RASQSLGSSYLA (SEQ ID NO: 7) LCDR2 GASSRAP (SEQ ID NO: 8) LCDR3 QQYADSPIT (SEQ ID NO: 9)

[0046] In some embodiments, the anti-TGFβ antibody is fresolimumab or the V H and / or V L In other words, the antibody comprises one or both of the following sequences: V H : QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SNVISWVRQA PGQGLEWMGG VIPIVDIANY AQRFKGRVTI TADESTSTTY MELSSLRSED TAVYYCASTL GLVLDAMDYW GQGTLVTVSS (SEQ ID NO: 10) V L : ETVLTQSPGT LSLSPGERAT LSCRASQSLG SSYLAWYQQK PGQAPRLLIY GASSRAPGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYADSPITFG QGTRLEIK (SEQ ID NO:11)

[0047] In some embodiments, the anti-TGFβ antibody is of the human IgG isotype, such as the human IgG4 isotype. In certain embodiments, the human IgG4 constant region comprises the following amino acid sequence:

[0048] [ka] In further embodiments, the human IgG4 constant region has a mutation at position 228 (Eu numbering). In some embodiments (e.g., Ab1), the mutation is a serine to proline mutation (S228P). In the above sequence, S228 serine is boxed.

[0049] In some embodiments, the anti-TGFβ antibodies (e.g., Ab1 and fresolimumab) comprise a human kappa light chain constant region (Cκ). In certain embodiments, the human Cκ comprises the following amino acid sequence: RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC (SEQ ID NO:13)

[0050] In some embodiments, antigen-binding fragments of complete anti-TGFβ antibodies can also be used. The term "antigen-binding fragment" or similar terms refers to a portion of an antibody that contains amino acid residues that interact with an antigen and confer to the binder its specificity and affinity for that antigen. Non-limiting examples of antigen-binding fragments include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single chain Fvs (scFvs), dAb fragments, and minimal recognition units composed of amino acid residues that mimic the hypervariable domains of an antibody.

[0051] In some embodiments, the antibody or antigen-binding fragment herein is linked to a bone-targeting moiety. In further embodiments, the bone-targeting moiety is a polyarginine (poly D) peptide. As used herein, the term "poly D peptide" refers to a peptide sequence having multiple aspartic acid or aspartate or "D" amino acids, such as about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or more aspartic acid amino acids (residues). For example, a poly D peptide can contain about 2 to about 30, or about 3 to about 15, or about 4 to about 12, or about 5 to about 10, or about 6 to about 8, or about 7 to about 9, or about 8 to about 10, or about 9 to about 11, or about 12 to about 14 aspartic acid residues. A poly D peptide may contain only aspartic acid residues or may contain one or more other amino acids or similar compounds. As used herein, the term "D10" refers to a contiguous sequence of 10 aspartic amino acids, as found in SEQ ID NO: 14. In some embodiments, an antibody or antibody fragment of the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 poly-D peptides.

[0052] The poly-D peptide can be connected to the anti-TGFβ antibody or antigen-binding fragment by fusion via recombinant technology, such that the poly-D is connected to the antibody or fragment through a peptidyl bond (i.e., the antibody or fragment is a fusion protein). For example, the poly-D peptide can be fused to the N-terminus or C-terminus, or both, of the heavy chain, and / or to the N-terminus or C-terminus, or both, of the light chain. The poly-D peptide can also be connected to the anti-TGFβ antibody or antigen-binding fragment by chemical conjugation, for example, by chemical reaction with a cysteine ​​or lysine residue on the antibody or antibody-binding fragment, with or without a linker moiety (e.g., maleimide functional group and polyethylene glycol (PEG)). See, for example, WO2018 / 136698.

[0053] In certain embodiments, the antibody is fresolimumab fused to D10 peptide at the N-terminus, C-terminus, or both termini of the heavy chain. In some embodiments, the antibody is fresolimumab fused to D10 peptide at the C-terminus of the light chain. In certain embodiments, the antibody is fresolimumab fused to D10 peptide at both termini of the heavy chain and at the C-terminus of the light chain.

[0054] In certain embodiments, the antibody is Ab1 fused to D10 peptide at the N-terminus, C-terminus, or both termini of the heavy chain. In some embodiments, the antibody is Ab1 fused to D10 peptide at the C-terminus of the light chain. In certain embodiments, the antibody is Ab1 fused to D10 peptide at both termini of the heavy chain and at the C-terminus of the light chain.

[0055] The anti-TGFβ antibody or antigen-binding fragment thereof of the present disclosure can be produced by methods well established in the art. The DNA sequences encoding the heavy and light chains of the antibody can be inserted into an expression vector such that the genes are operably linked to the necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The antibody light chain coding sequence and the antibody heavy chain coding sequence may be inserted into separate vectors and may be operably linked to the same or different expression control sequences (e.g., promoters). The expression vector encoding the antibody of the present disclosure is introduced into a host cell for expression. The host cell is cultured under conditions suitable for expression of the antibody, which is then recovered and isolated. The host cell includes mammalian, plant, bacterial, or yeast host cells. Mammalian cell lines available as hosts for expression include many immortalized cell lines that are well known in the art and available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and several other cell lines. Cell lines can be selected based on their expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells or Sf21 cells. Tissue culture media for the host cells may or may not contain animal-derived components (ADCs) such as bovine serum albumin. In some embodiments, culture media without ADCs is preferred for human safety. Tissue culture can be performed using fed-batch, continuous perfusion, or any other method appropriate for the host cells and the desired yield.

[0056] III. Pharmaceutical Compositions and Uses The methods described herein include administering to an OI patient a therapeutically effective amount of an anti-TGFβ antibody or antigen-binding fragment thereof. As used herein, the phrase "therapeutically effective amount" refers to a dose of an antibody that binds TGFβ that results in a detectable improvement in one or more symptoms associated with moderate to severe OI (e.g., type IV OI) or a biological effect (e.g., a reduction in the level of a particular biomarker) that correlates with an underlying pathological mechanism causing the condition or symptoms of moderate to severe OI.

[0057] Improved OI can be manifested as a decrease in bone turnover, a slower rate of bone remodeling, and / or a decrease in bone cell density. In some embodiments, improved OI is indicated by an improvement in a bone parameter selected from the group consisting of bone mineral density (BMD), bone mass density (BV / TV), total bone surface (BS), bone surface density (BS / BV), trabecular number (Tb.N), trabecular width (Tb.Th), trabecular center distance (Tb.Sp), and total bone mass (Dens TV).

[0058] In certain embodiments, the improved bone parameter is lumbar spine areal BMD (LS aBMD) determined by dual energy X-ray absorptiometry.Compared to the baseline level before treatment, the LS aBMD value can be improved by at least 1%, for example at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 150, 20, or more percent.

[0059] In some embodiments, BMD, bone mass, and / or bone strength is improved by about 5% to about 200% after treatment with a therapeutically effective amount of an anti-TGFβ antibody or fragment. In certain embodiments, BMD, bone mass, and / or bone strength is improved by about 5% to about 10%, 10% to about 15%, 15% to about 20%, 20% to about 25%, 25% to about 30%, 30% to about 35%, 35% to about 40%, 40% to about 45%, 45% to about 50%, 50% to about 55%, 55% to about 60%, 60% to about 65%, 65% to about 70%, 70% to about 75%, 75% to about 80%, 80% to about 85%, 85% to about 90%, 90% to about 95%, 95% to about 100%, 100% to about 105%, or 100% to about 105% after treatment. %, 105% to about 110%, 110% to about 115%, 115% to about 120%, 120% to about 125%, 125% to about 130%, 130% to about 135%, 135% to about 140%, 140% to about 145%, 145% to about 150%, 150% to about 155%, 155% to about 160%, 160% to about 165%, 165% to about 170%, 170% to about 175%, 175% to about 180%, 180% to about 185%, 185% to about 190%, 190% to about 195%, or 195% to about 200%.

[0060] In some embodiments, a therapeutically effective amount can result in a reduction in bone turnover, as indicated by a reduction in serum or urinary biomarkers such as, for example, urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type I cross-linked N-telopeptide (NTX), urinary or serum collagen type I cross-linked C-terminal-telopeptide (CTX), bone sialoprotein (BSP), osteopontin (OPN), and tartrate-resistant acid phosphatase 5b (TRAP). In certain embodiments, the reduction following treatment with an antibody that binds TGFβ is only about 5% to about 200% compared to baseline levels (e.g., before treatment). For example, the decrease may be from about 5% to about 10%, 10% to about 15%, 15% to about 20%, 20% to about 25%, 25% to about 30%, 30% to about 35%, 35% to about 40%, 40% to about 45%, 45% to about 50%, 50% to about 55%, 55% to about 60%, 60% to about 65%, 65% to about 70%, 70% to about 75%, 75% to about 80%, 80% to about 85%, 85% to about 90%, 90% to about 95%, 95% to about 100%, 100% to about 105%, 105% to about 110%, 120% to about 130%, 130% to about 140%, 140% to about 150%, 150% to about 160%, 160% to about 170%, 170% to about 180%, 180% to about 190%, 190% to about 200%, 200% to about 210%, 210% to about 220%, 220% to about 230%, 230% to about 240%, 240% to about 250%, 250% to about 260%, 260% to about 270%, 270% to about 280%, 280% to about 290%, 290% to about 300%, 300% to about 310%, 310% to about 320%, 320% to about 330%, 330% to about 340%, 340% to about 350%, 350% to about 400%, It can be 10% to about 115%, 115% to about 120%, 120% to about 125%, 125% to about 130%, 130% to about 135%, 135% to about 140%, 140% to about 145%, 145% to about 150%, 150% to about 155%, 155% to about 160%, 160% to about 165%, 165% to about 170%, 170% to about 175%, 175% to about 180%, 180% to about 185%, 185% to about 190%, 190% to about 195%, or 195% to about 200%.

[0061] In some embodiments, a therapeutically effective amount can result in an improvement in the levels of serum or urinary biomarkers of bone deposition, such as total alkaline phosphatase, bone-specific alkaline phosphatase, osteocalcin (OCN), and type I procollagen (C-terminal / N-terminal). In certain embodiments, the improvement is only about 5% to about 200% after treatment compared to baseline levels (e.g., before treatment). For example, improvements may be seen after treatment of about 5% to about 10%, 10% to about 15%, 15% to about 20%, 20% to about 25%, 25% to about 30%, 30% to about 35%, 35% to about 40%, 40% to about 45%, 45% to about 50%, 50% to about 55%, 55% to about 60%, 60% to about 65%, 65% to about 70%, 70% to about 75%, 75% to about 80%, 80% to about 85%, 85% to about 90%, 90% to about 95%, 95% to about 100%, 100% to about 105%, 105% to about 110%, 120% to about 130%, 130% to about 140%, 140% to about 150%, 150% to about 160%, 160% to about 170%, 170% to about 180%, 180% to about 190%, 190% to about 200%, 200% to about 210%, 210% to about 220%, 220% to about 230%, 230% to about 240%, 240% to about 250%, 250% to about 260%, 260% to about 270%, 270% to about 280%, 280% to about 290%, 290% to about 300%, 300% to about 310%, 310% to about 320%, 320% to about 330%, 330% to about 340%, 340% to about 350%, 350% to about 40 It can be 10% to about 115%, 115% to about 120%, 120% to about 125%, 125% to about 130%, 130% to about 135%, 135% to about 140%, 140% to about 145%, 145% to about 150%, 150% to about 155%, 155% to about 160%, 160% to about 165%, 165% to about 170%, 170% to about 175%, 175% to about 180%, 180% to about 185%, 185% to about 190%, 190% to about 195%, or 195% to about 200%.

[0062] In some embodiments, a therapeutically effective amount promotes bone deposition, hi some embodiments, a therapeutically effective amount improves the function of non-skeletal organs affected by OI, such as hearing, vision, pulmonary function, and renal function.

[0063] The therapeutically effective amount can be 1-10 mg / kg, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. In some embodiments, OI patients are treated with this amount of fresolimumab or Ab1 by intravenous injection. Treatment can be repeated at intervals deemed appropriate by the physician for the patient. In some embodiments, treatment with the anti-TGFβ antibody or antigen-binding fragment thereof can be repeated monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 9 months, every 12 months, or every 18 months.

[0064] The patient can be an adult (e.g., a patient aged 18 years or older). The patient can be a pediatric patient (a patient younger than 18 years, e.g., a patient between the ages of newborn and 6 years, between the ages of 6 and 12 years, or between the ages of 12 and 18 years).

[0065] IV. Combination Therapy In some embodiments, the anti-TGFβ antibody therapy of the present disclosure can be combined with other OI treatments. Examples of additional therapeutic agents include, but are not limited to, bisphosphonates, calcitonin, teriparatide, and any other compounds known to treat, prevent, or improve OI. Additional therapeutic agents can be administered simultaneously or sequentially with the antibody that binds to TGFβ. Examples of bisphosphonates are etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, zoledronate, and risedronate. In some embodiments, additional therapeutic agents are drugs that stimulate bone formation, such as parathyroid hormone analogs and calcitonin.

[0066] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in practicing or testing this disclosure. In the event of a conflict, the present specification, including definitions, shall control. Generally, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly practiced in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Throughout the specification and embodiments, the use of the words "have" and "comprise" or variations such as "has," "having," "comprises," or "comprising" are to be construed as implying the inclusion of a recited integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated herein by reference in their entirety. Although several documents are cited herein, such citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0067] In order that this invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. EXAMPLES

[0068] In the following examples, histology and RNA-seq were performed on bones from children affected (n=10) and unaffected (n=4) with OI. Gene Ontology (GO) enrichment assays, gene set enrichment assays (GSEA), and Ingenuity Pathway Analysis (IPA) were used to identify key dysregulated pathways and regulators. Reverse phase protein arrays (RPPA), Western blots (WB), and immunohistochemistry (IHC) were performed to confirm changes at the protein level. A phase I study of a single dose of fresolimumab, a pan-anti-TGFβ neutralizing antibody, at 1 or 4 mg / kg dose was performed in eight adults with OI type III and type IV. The safety of fresolimumab and the effect of fresolimumab on lumbar spine areal bone mineral density (LS aBMD) and bone remodeling markers were evaluated. Details of the materials and methods for the study are as follows.

[0069] Collection and processing of human bone samples Bones from children with and without OI were obtained under a protocol approved by the Institutional Review Board (IRB) of Baylor College of Medicine (BCM), Houston, TX, USA. Bone samples were obtained from children who had already undergone surgery for medical reasons. Bone fragments removed during surgery that would have otherwise been discarded were collected and processed. Informed consent was obtained from parents or legal guardians prior to collection of all samples. Following a previously reported protocol (Chou et al., Osteoarthritis Cartilage (2013) 21(3):450-61), bone specimens were processed in a liquid nitrogen-based environment as described in Figure 1.

[0070] RNA extraction, RNA-Seq, validation and data analysis Total RNA from pulverized bone was extracted using TRIzol® (ThermoFisher Scientific) and further purified by lithium chloride precipitation. RNA quality and quantity were measured by Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA was then subjected to RNA-seq, followed by validation and bioinformatics analysis of pathways and upstream regulators.

[0071] Protein extraction, reverse phase protein array (RPPA), Western blotting (WB), and immunohistochemistry (IHC) Proteins were extracted from pulverized bones overnight at 4°C by using lysis buffer (250 mM EDTA, 6 M guanidine-HCl, 50 mM Tris-HCl, pH 7.4). Extracts were concentrated by methanol-water-chloroform precipitation and dissolved in 4% SDS buffer (4SB; 4% SDS, 50 mM Tris, 5 mM EDTA, pH 7.4) for WB or diluted to 0.5 mg / ml in SDS sample buffer for RPPA. In total, three control bone samples and five OI type III bone samples were included in WB; four control bone samples and eight OI type III bone samples were included in RPPA and IHC (Table 1).

[0072] [Table 1]

[0073] Phase I Clinical Trial Design A phase I dose-escalation trial evaluating fresolimumab in adults with moderate-to-severe OI was conducted as part of the National Institutes of Health's Rare Disease Clinical Research Network's Brittle Bone Disorders Consortium. Stage 1 of the study involved a single infusion of fresolimumab (1 mg / kg body weight and 4 mg / kg body weight; n=4 in each dose cohort). Total follow-up was 6 months. The primary outcome measure was the safety of a single dose of fresolimumab. Secondary outcomes were to evaluate the effect of fresolimumab on lumbar spine areal bone mineral density (LS aBMD) by dual-energy x-ray absorptiometry (DXA) and circulating bone turnover markers (Ocn and CTX).

[0074] Individuals over 18 years of age with a diagnosis of moderate to severe OI based on 20 or more fractures and having a glycine substitution mutation in COL1A1 or COL1A2 or a biallelic pathogenic variant in CRTAP, PPIB, or LEPRE1 were enrolled.

[0075] Exclusion criteria were: 1) instrumentation of the LS and both hips that would prevent assessment of aBMD, 2) fracture of a long bone within 3 months prior to screening, 3) treatment with oral BPN within 6 months prior to screening or treatment with intravenous BPN and teriparatide within 12 months prior to screening, 4) scheduled skeletal surgery, and 5) features that may affect safety such as autoimmune disease, tuberculosis, history of cancer or precancerous lesions, valvular heart disease, or bleeding tendency. Markers of bone turnover were measured by a CLIA- and CAP-certified laboratory. DXA scans were performed at Texas Children's Hospital using a validated clinical machine. Scans were read by two central readers blinded to study procedures.

[0076] Histology and bone cell density analysis The treated bone specimens were then resuspended in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 48 h and decalcified in 10% EDTA (Sigma-Aldrich) for 14 days at 4 °C. Paraffin sections were stained with hematoxylin and eosin for morphological and osteocyte number analysis. Osteocyte density was calculated using BIOQUANT OSTEO (BIOQUANT Image Analysis Corporation, Nashville, TN, USA).

[0077] RNA-Seq and data analysis For RNA-Seq, 250 ng of total RNA was used for TureSeq Stranded mRNA library preparation (Illumina, San Diego, CA, USA) with ERCC spike-in (ThermoFisher Scientific) applied according to the manufacturer's instructions. 22 pM of equimolar pooled libraries were loaded into one lane of a high output v4 flow cell for bridge amplification using an Illumina cBot machine. Flow cells were sequenced on a HiSeq 2500 Sequencing System in High Output Mode with v4 chemistry (FC-401-4003, Illumina) using a paired-end 100-cycle run. PhiX Control v3 adaptor-ligated libraries (Illumina) were added at 2 wt% to ensure balanced diversity and to monitor clustering and sequencing performance. An average of 42.5 million paired-end reads were generated for each sample. Alignment was performed using HISAT2 by Genialis (https: / / www.genialis.com) with hg19-ERCC as reference. Then, normalization, differential expression, hierarchical clustering, and Gene Ontology analysis were performed using the RNA-seq analysis pipeline of Partek® Genomics Suite (Partek, St. Louis, MO, USA). Statistical significance was determined by ANOVA built into the Partek® Genomics Suite RNA-seq analysis pipeline. Significantly differentially expressed genes (fold change >2 and false discovery rate <0.05) were then loaded into Ingenuity® Pathway Analysis (IPA) (Qiagen, Hilden, Germany) for upstream regulator prediction. Gene Set Enrichment Analysis (GSEA) program for pathway enrichment analysis (Broad Institute) was used according to the developer's instructions.

[0078] RNA-seq validation with NanoString® A NanoString (Seattle, WA, USA) human WNT nCounter panel was run using one OI type III sample and one control sample with RNA-seq data for validation of detected gene expression fold changes. Analysis was performed using nSolver™ in the regular module. Genes with read counts below 20 were considered background and excluded from further analysis. A total of 155 genes were validated. Concordance was determined by the percentage of genes that showed the same change in expression direction.

[0079] Reverse Phase Protein Array (RPPA) RPPA was performed by using a standardized protocol (Marini et al., ibid.). For each sample, three technical replicates were assayed to account for technical variations. The protein expression intensity (PEI) of each biological sample was first derived from the calculation of the mean intensity of the technical triplicates of each biological sample. The mean expression intensity of the control or type III OI group was then calculated from the mean of the PEI of each sample within the group. The Student's t-test was used to determine statistical significance between control and type III OI. A nominal P-value of 0.05 was used to determine significance.

[0080] Western blotting (WB) 50 μg of total protein was used for loading. After separation by SDS-PAGE gel and transfer to PVDF membrane (MilliporeSigma, Burlington, MA, USA), the membrane was blocked using 5% milk, followed by incubation with primary antibodies (phospho-SMAD2 (3108S, Cell Signaling), SMAD2 (5339S, Cell Signaling)) and GAPDH (G9295, Sigma-Aldrich) at 4°C overnight. After appropriate secondary antibodies (Bio-Rad), the signal was captured using the ChemiDoc™ Gel Imaging System (Bio-Rad, Hercules, CA, USA).

[0081] Immunohistochemistry (IHC) After deparaffinization, sections were incubated with 0.05% trypsin at 37°C for antigen retrieval followed by 3% hydrogen peroxide treatment. After blocking with 5% normal goat serum, sections were incubated with primary antibody for phospho-SMAD2 (44-244G, ThermoFisher Scientific, Waltham, MA, USA) overnight at 4°C according to the manufacturer's instructions. Anti-rabbit secondary antibody (Vectastain ABC system, Vector Laboratories, Servion, Switzerland) was applied and blots were developed using 0.1% 3,3'-diaminobenzidine. EXAMPLES

[0082] Disorganized woven bone and increased osteocyte density in bones from children with type III of OI Bone fragments from the tibia or femur were collected from 10 children with type III OI (9 with glycine substitution mutations in COL1A1 or COL1A2 and 1 with a valine deletion in COL1A2) and 4 children not affected by OI (Table 1). Histologically, control specimens contained mainly cortical bone, whereas OI specimens contained both cortical and trabecular bone. Morphological examination revealed that OI bones manifested a disorganized Haversian system with predominantly fibrous bone compared to controls (Figure 2). Consistent with a previous report (Nijhuis et al., J Child Orthopaed. (2019) 13(1):1-11), osteocyte density was more than three times higher in OI bones (686.44 / mm in OI). 2 221.94 / mm vs. control 2 ), and compared to the control, the pits appeared more spherical (Figures 2 and 3). These data, typical for OI, imply the high quality of the samples. EXAMPLES

[0083] Global transcriptome analysis demonstrates elevated TGFβ signaling as a key dysregulated pathway in type III bone in OI To identify key dysregulated pathways in human OI bones in an unbiased manner, we performed RNA-seq using control and OI type III bones. To ensure the accuracy of the RNA-seq results, we validated 155 genes by NanoString® and demonstrated 92% concordance in expression fold change (Figure 4). Principal component analysis (PCA) of the transcriptome data revealed a clear separation between control and OI bones (Figure 5, Panel A). Gene expression profiles were more homogenous in controls than in OI bones. Hierarchical clustering of whole transcriptome RPKM showed distinct clusters between control and OI bones, indicating an altered global molecular signature in OI (Figure 5, Panel B). Following differential gene expression analysis, GO enrichment analysis was performed. In GO biological processes, significantly enriched skeletal-related functions captured the molecular signature of OI, including increased bone formation, bone resorption, and bone remodeling, along with decreased bone trabecular bone and bone maturation. Consistently, in GO cellular processes, heightened osteoblast and osteoclast differentiation was identified in OI. In GO signaling pathways, major bone remodeling pathways were upregulated, including BMP-TGFβ, parathyroid hormone pathway, WNT, and Notch signaling. Most interestingly, phosphorylation regulation of SMAD proteins was the most significantly upregulated pathway among all pathways (P=1.78×10 -15 ) (Figure 6). These enrichment results were not only consistent with the known molecular pathological features of OI bone, but also revealed without prejudice that SMAD phosphorylation, a downstream event of TGFβ, is the most affected target in type III bone with OI.

[0084] To independently explore signaling changes in OI bone, we next performed GSEA (Subramanian et al., PNAS (2005) 102(43):15545-50) using RPKM from control and OI bone. GSEA identified TGFβ signaling as the top pathway significantly activated in OI (Figure 5, panel C; Figure 7). Collectively, these results suggested that TGFβ signaling is the major and most consistently activated pathway in OI type III bone. EXAMPLES

[0085] TGFβ is an active upstream regulator in bone from type III of OI To investigate whether the activation of the TGFβ pathway identified from the transcriptome analysis was indeed due to TGFβ ligands, we used IPA to predict upstream regulators based on 3,722 significantly changed genes. Among all potential upstream regulators, TGFβ was the most activated upstream regulator identified (Z score = 4.28, P = 1.32 × l0 -14 ) (Table 2). To confirm these transcriptomic findings that led to changes at the protein level, we performed targeted proteomics RPPA using proteins extracted from bone. Of the 230 proteins examined, 29 proteins showed nominal statistically significant intensity changes between OI and control bones (Figure 8). This analysis identified TGFβ as the upstream regulator most significantly activated by IPA (Z score = 2.17, P = 6.37 x 10-12) (Table 2).

[0086] [Table 2] EXAMPLES

[0087] Increased SMAD2 phosphorylation in type III bone of OI To definitively demonstrate elevated TGFβ signaling in situ in type III bones with OI, we performed IHC using an antibody against a TGFβ downstream target, phosphorylated SMAD2 (pSMAD2).

[0088] Compared to controls, we found a consistent increase in pSMAD2 staining in type III bones with OI (Figure 9, Panel A). Through WB using protein lysates from control and type III bones with OI, we observed an increase in pSMAD2 in all OI samples examined (Figure 9, Panel B). Further quantification demonstrated a significant increase in the pSMAD2 / total SMAD2 ratio in OI bone (Figure 9, Panel C). Taken together, these results suggested that TGFβ activation is a driving pathogenic mechanism in humans with OI. EXAMPLES

[0089] Fresolimumab as a therapeutic intervention in OI To interpret our findings, we performed a phase I clinical trial evaluating the safety of fresolimumab, a human IgG4κ monoclonal antibody that neutralizes all mammalian isoforms of TGFβ. Consistent with the preclinical and human data we have generated, we enrolled only individuals with clinically moderate to severe OI caused by glycine substitution mutations in COL1A1 or COL1A2, or biallelic pathogenic variants in CRTAP, PPIB, or LEPRE1. A total of eight individuals were enrolled (Table 3).

[0090] [Table 3]

[0091] Four received a single dose of fresolimumab at a dose of 1 mg / kg body weight and four received a single dose of fresolimumab at a dose of 4 mg / kg body weight. Treatment with fresolimumab was well tolerated. There were no serious adverse events (AEs) and no clinically significant laboratory changes were observed in both cohorts (Figure 10). In the 1 mg / kg cohort, only two AEs were classified as probably / probably related to the study drug: nausea after drug administration and epistaxis. In the 4 mg / kg cohort, seven AEs were classified as probably / probably related to the study drug: fatigue, headache, epistaxis, urinary occult blood, bleeding from skin scabs, and corrected QT interval 457 ms at day 180 in one participant.

[0092] Treatment with fresolimumab 1 mg / kg was associated with an increase in Ocn and CTX, markers of bone turnover. A peak increase in bone remodeling was observed between 30 and 90 days after treatment. Treatment with the 4 mg / kg dose was associated with a sustained decrease in Ocn starting at 30 days after treatment (Figure 11). The two median readings of LS aBMD (masked to the time of measurement) had a high degree of correlation (r=0.995) and agreement (Figure 12). Therefore, the average aBMD from the two readings was used in calculating the percentage change from baseline. At the 1 mg / kg dose, two OI type IV participants showed a robust improvement in LS aBMD, while the OI VIII individual showed no change at all. The OI type III participant who showed a drop in aBMD had severe scoliosis that presented challenges, but the results from the DXA scan were analyzed.

[0093] In the 4 mg / kg cohort, LS aBMD was assessed at days 90 and 180. Two participants with OI type IV had robust improvements in aBMD by day 90. The individual with OI type III who showed a steep aBMD decline had sustained a femur fracture resulting in prolonged immobility; aBMD was measured at a remote facility, thus making the comparison less than ideal.

[0094] In conclusion, we have investigated in this study in a comprehensive and unbiased manner the global signaling abnormalities in OI, a Mendelian form of osteoporosis. The present findings and the "omic scale" data may not only have implications for the treatment of OI, but also be relevant for other disorders related to low bone mass. Furthermore, this study led to the surprising discovery that treatment with even a single dose of fresolimab was associated with changes in bone turnover and aBMD at days 90 and 180 in our study. In addition, the unique properties of bone biology provide a potential safety advantage, since a lower cumulative dosage and administration frequency would allow for reduced systemic toxicity. Indeed, the dose of fresolimumab administered herein was lower compared to the dose given in studies against melanoma, idiopathic pulmonary fibrosis, systemic sclerosis, and focal segmental glomerulosclerosis. No serious AEs were observed with the administration of a single dose. Furthermore, we observed differential effects on bone turnover markers between the two tested doses. At the 1 mg / kg dose, fresolimumab was associated with a mild increase in bone remodeling. However, 4 mg / kg fresolimumab treatment led to a sustained inhibition of bone turnover as indicated by plasma Ocn levels. The effect on LS aBMD was more variable depending on disease severity. Two participants with type IV OI improved their LS aBMD by 6.8% and 8.6% with a single dose of 1 mg / kg. In the 4 mg / kg cohort, one participant had a 7.6% improvement and two showed improvements of 2.9% and 1.3% 3 months after infusion. These gains are higher compared to the anabolic agent teriparatide, which demonstrated a 2% gain at 6 months in individuals with mild but not severe OI, and comparable to monthly high-dose setrusumab, which was associated with 5.4% of LS aBMD gains over the 6-month study period in OI types III and IV (Eric et al., JBMR Plus (2021) 5(S1):Suppl: e10455). Two participants with OI type III (FR005 and FR012) had a decline in aBMD.

[0095] Sequence Listing The table below shows the amino acid sequences referred to in this disclosure.

[0096]

Table 4

Claims

1. 1. Use of an anti-TGFβ antibody, or antigen-binding fragment thereof, in the manufacture of a medicament for use in a method of treating osteogenesis imperfecta (OI) in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-TGFβ antibody, or antigen-binding fragment thereof; the anti-TGFβ antibody or antigen-binding fragment thereof Heavy chain complementarity determining regions (CDRs) 1-3 comprising SEQ ID NOs: 4-6, respectively; and light chain CDR1-3 comprising SEQ ID NOs: 7-9, respectively; The therapeutically effective amount is 1 to 10 mg / kg.

2. The use according to claim 1 , wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain comprising SEQ ID NO:10 and a light chain variable domain comprising SEQ ID NO:

11.

3. The antibody is human IgG ４ The use according to claim 1 , comprising a constant region and / or a human kappa light chain constant region.

4. Human IgG ４ The use according to claim 3, wherein the constant region comprises the S228P mutation (Eu numbering).

5. The use according to claim 3 , wherein the antibody comprises a heavy chain comprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:

2.

6. The use according to claim 3 , wherein the antibody comprises a heavy chain comprising SEQ ID NO:3 and a light chain comprising SEQ ID NO:

2.

7. The use of claim 1 , wherein the antibody comprises a bone-targeting moiety, optionally wherein the bone-targeting moiety is a polyarginine peptide.

8. The use according to claim 7, wherein the antibody comprises one or more polyarginine peptides.

9. The antibody is an antibody or antigen-binding fragment thereof. at the N-terminus, or C-terminus, or both termini of the heavy chain, and / or At the C-terminus of the light chain, The use according to claim 8 , fused to a polyarginine peptide.

10. The use according to claim 7, wherein the polyarginine peptide is D10 (SEQ ID NO: 14).

11. The use according to any one of claims 1 to 10, wherein the OI is moderate to severe OI or type IV OI.

12. The use according to any one of claims 1 to 10, wherein the human subject is an adult patient (>=18 years) or a pediatric patient (<18 years).

13. The use according to any one of claims 1 to 10, wherein the human subject has a mutation in the COL1A1 or COL1A2 gene, optionally wherein the mutation is a glycine substitution mutation in the COL1A1 or COL1A2 gene, or a valine deletion in the COL1A2 gene.

14. 11. The use according to any one of claims 1 to 10, wherein the administration improves a bone parameter selected from the group consisting of bone mineral density (BMD), bone mass density (BV / TV), total bone surface (BS), bone surface density (BS / BV), trabecular number (Tb.N), trabecular width (Tb.Th), trabecular center distance (Tb.Sp), and total bone volume (Dens TV).

15. 15. The use of claim 14, wherein the bone parameter is lumbar spine area BMD (LS aBMD), and optionally LS aBMD is improved by at least 1-10% after administration compared to baseline levels.

16. 11. The use of any one of claims 1 to 10, wherein the administration reduces bone turnover and / or bone cell density, optionally wherein the reduced bone turnover is indicated by a reduced serum CTX or an increased serum osteocalcin (OCN).

17. The use according to any one of claims 1 to 10, wherein the therapeutically effective amount is 1 mg / kg.

18. The use according to any one of claims 1 to 10, wherein the therapeutically effective amount is 4 mg / kg.

19. The use according to any one of claims 1 to 10, wherein the administration is repeated every month, every two months, every three months, every six months, every nine months or every twelve months.

20. The use according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment is administered by intravenous infusion.

21. The use of any one of claims 1 to 10, wherein a bisphosphonate, parathyroid hormone, calcitonin, teriparatide, or an anti-sclerostin agent is also administered to the subject.

22. 22. The use according to claim 21, wherein the bisphosphonate is selected from alendronate, pamidronate, zoledronate and risedronate.

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