BETA VIA ITS COMPLEXES

RELATED PATENT APPLICATIONS

[0001 ] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/328,347 filed on April 27,2016. The entire content of the foregoing application is incorporated herein by reference, including all text, tables and drawings.

FIELD OF THE INVENTION

[0002] Embodiments of the invention relate to compositions, devices, systems and methods for selective removal of TGFp from blood or a blood product, and methods of using the same for treating a subject having, or suspected of having a disorder, symptom or disease.

INTRODUCTION

[0003] Transforming growth factor beta (TGF , TGFpi) is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes at least three different variant forms (i.e., TGFpl , TGF|32 and TGF|33). TGF|31 , TGF|32 and TGF|33 are highly pleiotropic cytokines that virtually all cell types secrete. TGFp molecules are proposed to act as cellular switches that regulate processes such as immune function, proliferation, and epithelial-mesenchymal transition. Targeted deletions of these genes in mice show that each isoform has some non-redundant functions. For example, TGFpl is involved in hematopoiesis and endothelial differentiation, TGF$2 affects development of cardiac, lung, craniofacial, limb, eye, ear, and urogenital systems, and TGF|33 influences palatogenesis and pulmonary development. However, TGF 1 , 2, and 3 have been found to be largely interchangeable in an inhibitory bioassay. TGFp knockout mice die around 4 weeks of age due to extensive inflammatory cell infiltrates.

[0004] TGFp is an immune-suppressive cytokine that is elevated systemically in cancer patients. TGFpl is also expressed by tumors as a mechanism to suppress immune cell activity. Presented herein, in some embodiments, are devices, compositions and methods for removing TGFp from blood or a blood product.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Fig. 1 shows a schematic of one embodiment of an apheresis approach. [0006] Fig. 2 shows a circulating plasma form of TGFp is the LLC (blue arrow) which is a therapeutic target for removal by apheresis. Since conventional antibodies to TGF do not recognize or bind to LLC, the binding agents, in some embodiments, bind specifically to the LLC for removal of this complex from the plasma of cancer patients.

[0007] Fig. 3 shows a diagram of an embodiment of a device with anti-TGF antibody conjugate beads. The device housing comprises glass with polypropylene frits and tips and caps. The materials are all Class 6 quality. All housing components are sterilized by EtO or purchased sterile. The beads comprising covalently linked anti-latent TGFpi are washed to remove uncoupled antibody, stored under bacteriostatic phosphate buffered saline with 0.9% benzyl alcohol (Bacteriostatic PBS).

[0008] Fig. 4 shows a bar graph illustrating amounts of TGFpi that were captured from latent TGFp complexes using the 7H4 antibody. Briefly, PBS/BSA was spiked with recombinant latent TGFp (R&D Systems) to 10 ng/mL. The solution was run through a 1 mL column bead bed coupled to 5 mg of antibody to Latent TGFp (7H4, Biolegend). The % capture of total TGFp at the successive indicated flow rates in imL/min was plotted. BSA- coupled beads were used as control ( p=0.0008[T-test, 2 tail, type 1 ]).

SUMMARY

[0009] In some aspects, presented herein is a method for extracorporeal depletion or removal of a transforming growth factor beta (TGFp) polypeptide, or complex thereof, from blood or a blood product comprising: a) contacting the blood or blood product with a solid surface comprising a binding agent that specifically binds to a TGFp polypeptide; and b) separating the blood or blood product from the solid surface. In some embodiments, step a) removes or depletes TGFp polypeptide from the blood or blood product, or reduces or decreases the amount of TGFp polypeptide present in the blood or blood product. In some embodiments, after c), the blood or blood product is introduced or re-introduced into a subject. In some embodiments, after c), the blood or blood product is introduced or reintroduced into the subject from which the blood or blood product was obtained. In some embodiments, prior to a) the blood or blood product is obtained from a subject.

[0010] In some embodiments, the subject is a mammal or human. In some embodiments the method of obtaining the blood or blood product from the subject and introducing the blood or blood product into the subject is by a process comprising apheresis or plasma exchange. In some embodiments, the solid substrate comprises a bead or particle and the binding agent is covalently linked to the bead or particle. In some embodiments, the solid substrate is housed in a column. In certain embodiments, the TGFp polypeptide comprises a TGFpi polypeptide. In some embodiments, the complex thereof comprises a small latent complex (SLC) comprising a TGFpi polypeptide, or a large latent complex (LLC) comprising a TGFpi polypeptide.

[001 1 ] In certain aspects, provided herein is a method of treating a subject having or suspected of having a disease or disorder comprising: a) contacting a blood or blood product obtained from the subject with a solid surface comprising a binding agent that specifically binds to a TGFp polypeptide or complex thereof; b) separating the blood or blood product from the solid surface and c) introducing the separated blood or blood product into the subject. In certain embodiments, the process of obtaining the blood or blood product from the subject and introducing the blood or blood product comprises apheresis or plasma exchange. In certain embodiments, step a) removes or depletes TGFp polypeptide from the blood or blood product, or reduces or decreases the amount of TGFp polypeptide present in the blood or blood product. In certain embodiments, the disease is a cancer, tumor or metastasis.

[0012] In some embodiments, the solid substrate comprises a bead or particle and the binding agent is covalently linked to the bead or particle. In some embodiments, the solid substrate is contained within a column.

[0013] In some embodiments, the binding agent is an antibody. In some embodiments, the binding agent comprises a monoclonal antibody, humanized monoclonal antibody, aptamer, camelid, X-aptamer, single chain antibody, TGFp receptor, or binding fragment thereof. In some embodiments, the binding agent binds to a small latent complex (SLC) comprising a TGFp polypeptide, a large latent complex (LLC) comprising a TGFp polypeptide or a latency-associated peptide (LAP) portion of TGFpi .

[0014] In certain embodiments, the TGFp polypeptide comprises a TGFpi , TGFp2 or TGFp3 polypeptide. In some embodiments, the complex thereof comprises a small latent complex (SLC) comprising a TGFp polypeptide, or a large latent complex (LLC) comprising a TGFp polypeptide.

[0015] In some aspects, presented herein is a device capable of removing a TGFp polypeptide, or complex thereof, from the blood or a blood product obtained from a subject, for example a human subject. In some embodiments, the device comprises a column containing a solid substrate comprising a binding agent that specifically binds to a TGFpi polypeptide, or complex thereof, wherein the column is configured for apheresis or plasma exchange. In some embodiments, the binding agent binds specifically to a LAP portion of a TGFp polypeptide. In some embodiments, the binding agent binds specifically to a small latent complex of TGFp or a large latent complex of TGFp. In certain embodiments, the device comprises a column, cylinder or cartridge having an inflow port and an outflow port, a solid surface comprised with the column, cylinder or cartridge, and the binding agent is covalently attached to the solid surface.

[0016] In some aspects, a device described herein is used to treat a subject having, or suspected of having a cancer, tumor or metastasis.

DETAILED DESCRIPTION

[0017] TGFp polypeptides (e.g., TGFpi , TGFp2, and TGFP3) are multifunctional secreted cytokines that play pivotal roles in diverse biological processes, including the regulation of cell growth and survival, cell and tissue differentiation, development, inflammation, immunity, hematopoiesis, and tissue remodeling and repair. For example TGFpi plays a significant role in cancer ^<1) _: is essential for wound healing, stimulates matrix molecule deposition and angiogenesis, and is an essential mediator of pathologic scarring in fibrotic disorders.

[0018] TGFpi , TGFp2, and TGFP3 are initially synthesized as precursor proteins that undergo proteolytic cleavage by a similar process to form latent complexes. For example, TGFpi is synthesized as a single propeptide precursor of 390-amino acids with an N- terminal signal peptide of 29 amino acids, a 249 amino acid pro-region (LAP), and a 1 12 amino acid C-terminal region (TGFp). Furin cleaves the protein at residue 278, yielding an N-terminal cleavage product which corresponds to the latency-associated peptide (LAP), and a 25-kD C-terminal portion. The disulfide linked homodimers of LAP and homodimers of the TGFp C-terminal portion remain non-covalently associated, forming the small latent TGFp complex (SLC, 100kD). This complex remains in the cell until it is bound by another protein called Latent TGF-p-Binding Protein (LTBP), forming a larger complex called Large Latent Complex (LLC, 235-260 kD). It is this LLC that gets secreted to the extracellular matrix (ECM). Once secreted, the TGFp homodimer can be activated and released from the LLC complex by a variety of different mechanisms. These activation mechanisms may involve proteases that degrade LAP, thrombospondin-1 , reactive oxygen species, and integrins avb6 and avb8, some of which are mechanisms are pH dependent.

[0019] Signaling begins with binding to a complex of the accessory receptor betaglycan (also known as Ri ll) and a type I I serine/threonine kinase receptor termed Rll. The active TGFp dimer signals by bringing together two pairs of receptor serine/threonine kinases known as the type I and type II receptors, respectively. On binding TGFp, the type II receptors phosphorylate and activate the type I receptors (either ALK-1 or Rl (also called ALK-5)) that then propagate the signal by phosphorylating Smad transcription factors.

Receptors of the TGFp branch of the cytokine family phosphorylate Smads 2 and 3, whereas those of the other branch such as BMP receptors phosphorylate Smads 1 , 5, and 8. Use of other signaling pathways that are Smad-independent allows for distinct actions observed in response to in different contexts. Once activated, the receptor substrate Smads (RSmads) shuttle to the nucleus and form a complex with Smad4, a binding partner common to all RSmads ^<2) . Through combinatorial interaction with different transcription factors, a common TGFp stimulus can activate or repress hundreds of target genes at once. Variant signaling branches and Smad-independent pathways coexist with the canonical Smad pathway in the response to TGFp. Seven type I receptors and five type II receptors paired in different combinations provide the receptor system for the entire TGFp family. Alterations and frame shifts are found at the level of the TGFp receptors in cancer.

[0020] As an immunosuppressive cytokine, TGFp inhibits the development, proliferation, and function of both the innate and the adaptive arms of the immune system. Targets of TGFp include CD4 ⁺ effector T cells (Th1 and Th2), CD8 ⁺ cytotoxic T cells (CTLs), dendritic cells, NK cells, and macrophages. Additionally, TGFp stimulates the generation of regulatory T cells (Treg), which inhibit effector T cell functions, and IL17-producing Th17 cells, which regulate NK cells and macrophages.

[0021 ] TGFp is a potent inducer of Epithelial-mesenchymal transition (EMT). Cells undergoing EMT lose expression of E-cadherin and other components of epithelial cell junctions. Instead, they produce a mesenchymal cell cytoskeleton and acquire motility and invasive properties. It has been have shown that TGFp in the tumor environment primes cells for metastasis through the angiopoietin-like 4 (ANGPTL4) expression pathway' ³ '. TGFp can also enhance cell motility by cooperating with HER2 signals, as observed in breast cancer cells overexpressing HER2. TGFp stimulates the generation of myofibroblasts from mesenchymal precursors' ⁴ '. Myofibroblasts have features of fibroblasts and smooth muscle cells and are highly motile. Their presence in tumor stroma, partly as what are called "cancer-associated fibroblasts," facilitates tumor development. Glioma cell cultures proliferate in response to TGFp through the induction of platelet-derived growth factor B (PDGF-B) through epigenetic processes.

[0022] Mutational inactivation of core pathway components occurs in large subsets of colorectal, pancreatic, ovarian, gastric, and head and neck carcinomas. Breast cancers, prostate cancers, gliomas, melanomas, and hematopoietic neoplasias preferentially disable the tumor-suppressive action of TGFp by losing the tumor-suppressive arm of the signaling pathway. In lung adenocarcinomas and squamous cell carcinomas the loss of the TGFBRII results in aggressive tumor growth and reduced survival' ⁵ '.

[0023] Elevated plasma levels of have been found for several cancer types including colorectal, prostate, bladder, breast, pancreatic, or renal cancers and on myeloma and lymphoma. High levels of TGFpi immunostaining in infiltrating breast carcinoma has long been associated with metastasis' ⁶ '. It is now generally accepted that high levels of TGFp in the tumor microenvironment influence primary tumor cells toward metastatic potential ^<3) . In support of this is ER ^" breast tumors, where low expression of TGFBRII is associated with a favorable outcome' ⁷ '. In mice, where TGFp levels are increased by radiation or

chemotherapy the use of blockers prevents lung metastasis' ⁸ '. Studies of the role of TGFp in murine metastases have shown various effects pro-metastatic or anti-metastatic depending on the context. The bottom line is that metastatic cells need to have motility and extravasation to seed distal tumors and this is associated with high levels of TGFp. In this regard, TGFp stimulates expression of ANGPTL4 which functions to disrupt vascular endothelial cell junctions, to increase permeability of lung capillary walls and facilitates seeding of pulmonary metastases.

[0024] In addition to the role of TGFp in local tumor invasion, growing evidence implicates TGFp in the promotion of distal metastasis. Metastasis proceeds through a series of steps whereby cancer cells enter the circulatory system, disseminate to distal capillary beds, enter a parenchyma by extravasation, adapt to the new host microenvironment, and eventually grow into lethal tumor colonies in those distal organs ' ^{9, 10)} . Metastasis follows characteristic organ distribution patterns that reflect distinct colonization aptitudes of cancer cells from different origins, different tumor-efferent circulation patterns, and distinct compatibilities between disseminated cells and the organ that they encounter. Beyond the proliferative, survival, and invasive functions of a malignant state, metastasis requires extravasation and colonization functions that come into play once malignant cells disseminate. Such functions may be acquired in the primary tumor but become selected mainly during seeding and colonization distal metastases. Studies in model systems have described a broad range of potential and sometimes contradictory TGFp effects on metastasis.

[0025] Bone metastases are a significant problem in late stage breast cancer patients. Following their mobilization into marrow, cancer cells trigger osteoclasts to release which further influences cytokine release which in turn enhances metastatic invasiveness' ¹¹ '. Two genes that modulate bone metastases in ER ^" breast cancer cells are interleukin- 11 (IL- 11) and connective tissue growth factor (CTGF). These are TGFp target genes. CTGF is an extracellular mediator of invasion and angiogenesis, whereas IL-1 1 stimulates the production of the osteoclastogenic factors RANKL and GM-CSF in osteoblasts. Induction of IL-11 and CTGF expression by TGFp is mediated by the Smad pathway' ¹² ' and has been confirmed in malignant cells isolated from patients with metastatic breast cancer ^<13) . TGFp also induces IL-10 and IL-6 expression where IL-10 provides positive feedback for TGFp expression. In ER ^" breast tumors that are positive for both the TGFp gene response signature and lung metastasis signature (LMS) are associated with the highest risk of relapse through lung metastases ^<14) . Patients with these signatures showing enhanced function may be selective candidates for TGFp blocking therapy.

[0026] With mounting clinical evidence that TGFp acts as a tumor-derived

immunosuppressor, an inducer of tumor mitogens, a promoter of carcinoma invasion, and a trigger of prometastatic cytokine secretion, there is increasing interest in TGFp as a therapeutic target. Blocking TGFp signaling by overexpressing the inhibitor Smad7 or a dominant-negative form of the TGFp receptor deters the formation of osteolytic metastases by human breast cancer' ¹⁵ ', melanoma' ¹⁶ ' and renal carcinoma cell line xenografts' ¹⁷ '. One of the mediators of TGFp osteolytic action is parathyroid hormone-related protein (PTHrP) ^{<11 )} . TGFp stimulates PTHrP secretion without appearing to increase PTHrP imRNA levels. PTHrP stimulates the production of RANK ligand (RANKL) in osteoblasts, which in turn promotes the differentiation of osteoclast precursors and bone resorption. Administration of anti-PTHrP neutralizing antibodies inhibits TGFp-dependent osteolytic bone metastasis in mice ^<18) . The role of TGFp in metastatic colony expansion may not be limited to bone metastasis. A majority of metastases to lung, liver, and brain in breast cancer patients stain positive for phospho-Smad2, suggesting a widespread activation of this pathway in metastasis by locally released TGFp. In breast cancer cells that have entered the pulmonary parenchyma, TGFp may facilitate tumor reinitiation through an aberrant induction of ID1 expression' ³ '.

[0027] Inhibitors of the TGFp pathway developed to date encompass several classes. They include antisense oligonucleotides, inhibitors of ligand-receptor interactions such as anti- TGFp antibodies' ¹⁹ ', anti-receptor antibodies, TGFp-trapping receptor ectodomain proteins, and small-molecule inhibitors that target TGFp receptor kinases. A few anti-TGFp compounds have shown efficacy in preclinical studies and several of these compounds are being evaluated in clinical trials ' ^{20, 21 )} . Clinical trials for each of these inhibitor classes have been initiated not only against cancers (glioma, melanoma, breast cancer) but also against fibrosis, scarring, and other conditions that result from excessive TGFp activity. Despite the potential that TGFp signaling might enhance the progression of premalignant lesions, offsetting potential clinical benefit exists especially for patients with TGFp promoted tumor growth. In spite of such concerns, systemic administration of TGFp blockers has not been reported to increase spontaneous tumor development in animal models with one possible exception. Prolonged treatment with an antibody (1 D1 1 ) that binds to all three isoforms of TGFp, was shown to give rise to carcinomas. Such lesions are believed to be derived from pre-malignant foci that normally remain suppressed in the absence of antibody. Indeed, when antibody treatment is discontinued, the carcinomas resolve.

[0028] Therapeutic targeting of the TGFp pathway in tumors such as glioma, melanoma, and renal cell carcinoma is based on the rationale that TGFp exerts strong

immunosuppressive effects in these tumors. Thus, blocking or inhibiting TGFp function might empower the immune system against tumors. Blocking or inhibiting TGFp action may also have additional tumor-specific benefits. For example, TGFp inhibition in gliomas may curtail the production of autocrine survival factors, such as PDGF. Blocking or inhibiting TGFp in ER ^" breast cancer, on the other hand, might prevent primary or metastatic tumors from seeding and reseeding metastasis' ²² '. Finally, in osteolytic bone metastasis, blocking or inhibiting TGFp might interrupt the cycle of TGFp-induced osteoclastogenic factors and halt tumor growth. Although these examples show the great potential of the pathway as a therapeutic target, there are potential negative consequences, as well. Inhibition of TGFp might lead to chronic inflammatory and autoimmune reactions, although this problem has not yet materialized in the preclinical or clinical trials of systemic TGFp blockers. Inhibition of TGFp receptor function might also lead to runaway compensatory mechanisms by other activators of the Smad pathway, similar to what occurs in individuals with inactivating mutations in TGFBRI or TGFBRII ^<23) .

[0029] Progress in delineating the protumorigenic effects and mechanisms of TGFp in specific tumor types and in different stages of cancer progression is essential for determining when and how anti-TGFp targeted therapy might be feasible. The recent development of TGFp gene expression prognostic tools and TGFp response biomarkers may provide the means to select patients for anti-TGFp intervention in addition to a way to assess effective pharmacological targeting of this pathway.

[0030] TGFp is an immunosuppressive cytokine that is elevated systemically in cancer patients. TGFpi is also expressed by tumors as a mechanism to suppress immune cell activity. Presented herein, in some embodiments, is an affinity column for the removal or depletion of TGFp from the plasma, blood or blood product using any apheresis systems. In some embodiments, presented herein is a device to specifically remove or deplete TGFp as a treatment strategy for cancer, tumor or metastasis. In certain embodiments, this is accomplished by covalently linking an anti-TGFp antibody to circulating TGFp as a capture ligand to a support matrix. In certain embodiments, patient plasma, blood or blood product is treated by placing a column (OEM components) containing the TGFp binding matrix into a plasma flow line of the apheresis system for plasma exchange to capture and thus remove or deplete the patient's TGFp. As the TGFp is removed or depleted, the systemic reservoir concentration of TGFp is reduced to desuppress anti-cancer effects with ramifications at the tumor site(s). The device performance is dependent on the efficiency of binding TGFp complexes by the immobilized binding agent (e.g., antibody). The stable covalent linkage of the capture binding agent (e.g., antibody) on the matrix prevents its release into the patients being treated.

[0031 ] Since TGFp is a suppressor of immune responses, transitory inhibition or removal of TGFp by apheresis may stimulate immune responses to tumor cells in cancer patients and may provide therapeutic benefit alone or in combination with other anti-cancer therapies. Presented herein, in some embodiments are affinity cartridges that can be used in any apheresis setting for the removal of specific proteins from patient plasma, blood or blood product during apheresis. Presented herein, in some embodiments are cartridges designed for the specific removal or depletion of TGFp from the plasma, blood or blood product of cancer patients for a therapeutic anti-cancer approach.

[0032] Systemic elevation of TGFp is common to cancers. The removal or depletion of TGFp by selective affinity based plasmapheresis, at least transiently, may be another alternative to induce anti-tumor effects. This method is a novel approach to treat cancer patients through transient potentiation and systemic release from the immune suppressive effects of TGFp. The approach is to remove or deplete TGFp using a blood filtration affinity column in conjunction with plasmapheresis (immunopheresis) to selectively capture circulating latent complexes from the plasma, blood or blood product. The TGFp depleted plasma, blood or blood product is returned to the patient. This in turn can stimulate antitumor and anti-metastatic responses to reduce tumor burden and may lead to protective anti-tumor immunity. The feasibility of subtractive targeting TGFp has several aspects that justify this approach:

1. Cancers have a high level of TGFp in the tumor microenvironment that is

immunosuppressive by stimulation of the following: b. IL-11

c. IL-6

2. The systemic administration of the anti- antibody 1 D1 1 that inhibits TGFpi , TGFp2 and TGFp3 was shown to enhance an anti-cancer vaccine' ²⁴ ' in preclinical studies.

3. Anti- TGFp approaches have potential for the clinic' ²⁰ ' ²¹ ' ²⁵⁾ .

[0033] Cancer therapeutic approaches involving the inhibition of immunosuppressive cytokines are considered to have advantages over chemotherapy, radiation as well as surgeries, the last because of the considerable number of inoperable tumors. Additionally, the modulation of immune response has the potential for lower side effects since restoration to homeostasis quickly follows short term inflammatory immune responses by mimicking natural scenarios. One candidate immune suppressor target is TGFp, a pleiotropic cytokine whose inhibition has been shown to promote anti-cancer effects. Use of an anti-TGF antibody, 1 D1 1 , resulted in the reduction of tumor burden as well as increasing the survival rates in small animal studies and displayed overall beneficial responses in human clinical trials. However the administration of anti-TGF antibody serves to inhibit TGFp activity but does not reduce the overall systemic levels of circulating TGFp.

[0034] There are several problems associated with antibody infusion therapy. The infusion of antibody is dependent on pharmacokinetic and pharmacodynamic factors that are dose and time dependent. This complicates the dosing requirements for both efficacy and safety considerations. To overcome the complications of therapeutic administration of anti-TGF antibody, the methods herein enable the ability to reduce the levels of circulating TGFp. One embodiment described herein is a method to remove circulating TGFp and thus control the overall systemic blood plasma concentration of the cytokine that is not readily achievable with antibody infusion. Additional robustness of treatment can be implemented since real time observations are complemented by the ability to immediately cease the cytokine removal process and then restart the process on demand. The use of a plasmapheresis system compatible with a TGFp capture device allows treatment without the infusion of therapeutic agents.

[0035] Since anti-TGF antibodies recognize mature un-complexed TGFp, their inhibitory activity to TGFp is dependent on the dissociation of the TGFp polypeptide from LAP. Thus, the infusion of anti-TGF antibodies is intended to inhibit the function of TGFp at sites where it is activated and released from the LTBP complex, for example at cellular receptor sites. Such TGFp activation achieved by proteases at the site of action as a prelude to ligation of

10 TGFp to its receptor. For in-vitro diagnostic assays, the dissociation of TGFp from the complex is achieved by an acidification step to lower the pH to approximately 2 followed by a neutralization step. The free TGFp protein can then be detected for instance, by an ELISA assay.

[0036] Others have developed a relatively specific adsorption for hydrophobic molecules, a technique that is dependent on hydrophobic surface interactions to remove TGFp complexes from the plasma. However, it is inherently non-specific for TGFp since a variety of different proteins are expected to non-specifically interact with hydrophobic surfaces. Instead of using non-selective hydrophobic surfaces, embodiments presented herein capture and deplete TGFp in patient plasma, blood or blood product using immobilized binding agents. Since circulating LAP-bound TGFp complexes are not generally recognized by anti-TGF antibodies due to masking of the antigenic sites, in certain methods disclosed herein uses monoclonal antibodies that recognize LAP that are linked to solid surfaces (e.g., affinity matrices) to selectively target and remove TGFp complexes. In some embodiments, the method makes use of extracorporeal plasmapheresis/affinity chromatography systems such as those used for commercial plasma exchange. The embodiment includes the use of a device that is compatible with such subtractive procedures for targeted reduction of systemic TGFp as a method for the treatment of cancer. In some embodiments, matrices are comprised in a column device through which patient plasma, blood or blood product is circulated such that the TGFp complex is captured on the matrix contained within the device. The matrix is coupled to a binding agent that is specific for capture of circulating TGFp complexes. The device outflow which is depleted of TGFp is then returned to the patient during the plasmapheresis or plasma exchange procedure. The resulting lowering of systemic levels of TGFp thus attenuates its immunosuppressive effects and activates anticancer, tumor or metastasis immune responses.

[0037] A TGFp polypeptide refers to polypeptide comprising an amino acid sequence of a mature TGFpi , a mature TGFp2, or mature TGFP3 peptide. The mature form of TGFpi , 2 and 3 is that portion of the molecule that can bind to, and active a TGFp receptor. In some embodiments a TGFp polypeptide comprises a TGFp leader sequence (e.g., a leader sequence of TGFpi , 2 or 3) and/or a TGFp LAP polypeptide (e.g., a LAP polypeptide of TGFpi , 2 or 3). A TGFp polypeptide may comprise any known naturally occurring variant of a TGFp polypeptide.

[0038] In some embodiments a TGFp polypeptide is a mammalian TGFp polypeptide. In some embodiments a TGFp polypeptide is a primate TGFp polypeptide. In some embodiments a TGFp polypeptide is a human TGFp polypeptide. In some embodiments a TGFp polypeptide is a monkey TGFp polypeptide. In some embodiments a TGFp polypeptide is a rodent TGFp polypeptide (e.g., rat and/or mouse). In some embodiments a TGFp polypeptide is a canine TGFp polypeptide (e.g., a dog TGFp polypeptide). Non- limiting examples of a human TGFp polypeptide are provided in Example 3 and/or in a sequence listing of this application. In certain embodiments, a TGFp polypeptide comprises TGFpi or a mature peptide of TGFpi . In certain embodiments, a TGFp polypeptide comprises TGFp2 or a mature peptide of TGFp2. In certain embodiments, a TGFp polypeptide comprises TGFp3 or a mature peptide of TGFp3. In some embodiments, a TGFp polypeptide comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90% or at least 95% to a TGFp polypeptide disclosed herein. In some embodiments, a TGFp polypeptide comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% to SEQ ID Nos: 1 , 2 or 3. In some embodiments, a TGFp polypeptide comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% to the mature TGFp peptide SEQ ID Nos: 1 , 2 or 3.

[0039] In some embodiments, a binding agent disclosed herein binds specifically to a complex of a TGFp polypeptide. In some embodiments, a complex of a TGFp polypeptide comprises a TGFp LAP polypeptide. In some embodiments, a complex of a TGFp polypeptide comprises a TGFp latency-associated peptide (LAP). In some embodiments, a complex of a TGFp polypeptide is a small latent TGFp complex (SLC). In some

embodiments, a complex of a TGFp polypeptide comprises a Latent TGF-p-Binding Protein (LTBP). In some embodiments, a complex of a TGFp polypeptide comprises a Latent TGF- β-Binding Protein (LTBP) and a LAP. In some embodiments, a complex of a TGFp polypeptide is a large latent TGFp complex (LLC). In certain embodiments, a complex of a TGFp polypeptide comprises a mature peptide of TGFpi , a LAP derived from TGFpi and/or an LTBP.

[0040] In certain embodiments, blood or a blood product comprises a TGFp polypeptide, or a complex thereof. In some embodiments, a method or device described herein removes a TGFp polypeptide, or complex thereof from blood or a blood product. In some

embodiments, a method or device described herein removes at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of a TGFp polypeptide, or a complex thereof, from the blood or blood product of a subject. In some embodiments, a method or device described herein reduces the amount of a TGFp polypeptide, or complex thereof, in the blood or a blood product obtained from a subject. The term "blood" refers to whole blood. Non-limiting examples of a blood product include serum and plasma.

[0041 ] Presented herein, in some embodiments, a method or device described herein is used for the treatment of a disease or disorder in a subject.

[0042] The term "subject" refers to a mammal. Any suitable mammal can be treated by a method or composition described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human, such as a human having a tumor, cancer or metastasis.

[0043] In some embodiments, a solid substrate or device described herein comprises one or more binding agents. In some embodiments a binding agent binds specifically to a TGFp polypeptide, a complex thereof (e.g., an SLC or LLC), a portion thereof, or a fragment thereof. In some embodiments a binding agent binds specifically to a LAP portion of a TGFp polypeptide. In some embodiments a binding agent binds specifically to an SLC complex. In some embodiments a binding agent binds specifically to TGFpi , or a complex thereof. In some embodiments a binding agent binds specifically to TGFp2, or a complex thereof. In some embodiments a binding agent binds specifically to TGFp3, or a complex thereof.

[0044] A binding agent sometimes comprises or consists of a suitable antibody, an antibody fragment and/or an antigen binding portion thereof (e.g., a binding fragment). In some embodiments a binding agent is an antibody or an antigen binding portion thereof. An antibody can refer to a natural antibody, polyclonal antibody, monoclonal antibody, non- naturally occurring antibody, recombinant antibody, chimeric antibody, and antibody binding fragments (e.g., an antigen binding portion of an antibody), a CDR-grafted antibody, a humanized antibody, a human antibody, or portions thereof. In some embodiments, a binding agent is a non-naturally occurring antibody, non-limiting examples of which include chimeric antibodies, recombinantly produced monoclonal antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv fragment, single-chain Fv (scFv), diabody (Dab), synbody, TandAbs, nanobodies, BiTEs, SMIPs, DARPins, DNLs, affibodies, camelid, Duocalins, adnectins, fynomers, Kunitz Domains Albu-dabs, DARTs, DVD-IG, Covx-bodies, peptibodies, scFv-lgs,

SVD-lgs, dAb-lgs, Knob-in-Holes, triomAbs, and the like. In certain embodiments, a binding agent is an aptamer. In certain embodiments, a binding agent is a peptide aptamer. In certain embodiments, a binding agent is an X-aptamer. In certain embodiments, a binding agent is a nucleic acid based aptamer. Any suitable method can be used to make a binding agent that specifically binds to a TGFp polypeptide or complex thereof.

[0045] In some embodiments, an antibody is derived, obtained, isolated, or purified from a suitable animal non-limiting examples of which include rabbit, goat, horse, ruminant (e.g., goats, sheep, giraffes, yaks, deer, antelope, cows and the like), rodent (rat, mouse, hamster), pig, fish, bird (e.g., chicken, e.g., bird eggs), llama, or the like. In some embodiments an antibody is derived, obtained, isolated, or purified from a suitable mammal. In certain embodiments a suitable mammal is a genetically altered mammal (e.g., a trans chromosomal or transgenic mammal) engineered to produce antibodies comprising human heavy chains and/or human light chains or portions thereof. In some embodiments, an antibody is derived, obtained, isolated, or purified from a rabbit, goat, horse, cow, rat, mouse, fish, bird, or llama, for example.

[0046] A binding agent that specifically binds to a TGFp polypeptide, or complex thereof, can be made by any suitable method. Methods for generating antibodies, recombinant antibodies and/or antigen binding portions thereof are known. In some embodiments a binding agent is obtained from a suitable expression library. In some embodiments, a monoclonal binding agent is isolated from a phage library of binding agents, for example by using a technique described in Clackson et al, Nature, 352:624-628 (1991 ) and/or Marks et al, J. Mol Biol, 222:581 -597 (1991 ), or a variation thereof. The genes, or portions thereof, that encode a polypeptide of a binding agent may be cloned, subcloned, rearranged or modified for recombinant expression by a suitable cloning procedure and subsequently expressed using a suitable expression system by a method known to those skilled in the art (e.g., see Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982; Antibody Engineering: Methods and Protocols, Vol. 248 of Methods in molecular biology, edited by Benny K. C. Lo, Springer Science & Business Media, 2004; Antibody Engineering, Vol. 1 , Roland E. Kontermann, Stefan Dubel, Edition 2, Publisher Springer Science & Business Media, 2010; Antibody Phage Display: Methods and Protocols, Biomed Protocols, Vol. 178 of Methods in molecular biology, Editors Philippa M. O'Brien, Robert Aitken, Springer Science & Business Media, 2004; which are hereby incorporated by reference in their entirety). Methods for generating chimeric, grafted and/or humanized antibodies are known (see, e.g., U.S. patent No. 5,530,101 , U.S. patent No. 5,707,622, U.S. patent No. 5,994,524 and U.S. patent No. 6,245,894), which generally involve exchanging an antibody variable region, or portion thereof, from one species (e.g., mouse) into an antibody constant domain of another species (e.g., human). In some embodiments, an antibody can be humanized by exchanging one or more framework regions, or portions thereof (e.g., one or more individual amino acids), with one or more framework regions, or portions thereof (e.g., one or more individual amino acids), from a human antibody. Methods of humanizing an antibody by transferring one or more CDRs (e.g., 1 , 2, 3, 4, 5 or all 6 CDRs) from a donor binding agent (e.g., a binding agent comprising framework regions of a mouse monoclonal antibody) to an acceptor binding agent (e.g., a binding agent comprising human framework regions) while retaining antigen binding are known (e.g., see Queen et al., (1988) PNAS 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327; Antibody Engineering: Methods and Protocols, Vol. 248 of Methods in molecular biology, edited by Benny K. C. Lo, Springer Science & Business Media, (2004) ; Antibody Engineering, Vol. 1 , Roland E. Kontermann, Stefan Dubel, Edition 2, Publisher Springer Science & Business Media, (2010)). According to certain embodiments a binding agent is modified to include certain amino acid additions, substitutions, or deletions designed to (1 ) reduce susceptibility of a binding agent to proteolysis, (2) reduce susceptibility of a binding agent to oxidation, (3) alter binding affinity to Fc receptors, (4) alter antigen binding affinity of a binding agent, (4) increase serum half-life and/or (5) confer or modify other physicochemical, pharmacokinetic or functional properties of a binding agent.

[0047] A binding agent can be expressed, isolated from and/or purified from a suitable expression system non-limiting examples of which include a suitable bacteria, insect, plant or mammalian expression system. For example, a nucleic acid encoding a binding agent can be introduced into a suitable mammalian cell line that expresses and secretes the binding agent into the cell culture media.

[0048] In certain embodiments, a binding agent comprises a TGFp receptor (e.g., TGFp Receptor 1 , or TGFp Receptor 2), or a TGFp binding portion thereof.

[0049] The term "specifically binds" refers to a binding agent that binds to a TGFp polypeptide, or complex thereof, in preference to binding other molecules or other peptides as determined by, for example, a suitable in vitro assay (e.g., an Elisa, Immunoblot, Flow cytometry, and the like). A specific binding interaction discriminates over non-specific binding interactions by about 2-fold or more, often about 10-fold or more, and sometimes about 100-fold or more, 1000-fold or more, 10,000- fold or more, 100,000-fold or more, or 1 ,000,000-fold or more. [0050] Non-limiting examples of anti-TNFp antibodies that can be used for a method or device described herein include TW7-28G11 (Biolegend), and clone 7H4 (Biolegend), which bind to LLC.

[0051 ] In certain embodiments, blood or a blood product is contacted with a solid surface comprising a binding agent and the blood or blood product is then separated from the solid surface. A solid surface may comprise an insoluble organic or inorganic solid that may be particulate, crystalline, polymeric, fibrous, porous-hollow fibrous, monolithic, or

membranaceus in nature. Non-limiting examples of a solid surface include beads, particles, fibers, porous-membranes, porous-walled hollow fibers, and monolithic structures (a monolith), including combinations of multiple structural types. A solid surface may further comprise or consist of compound constructions, for example in which particles are embedded in a reticular matrix, sandwiched between membranes, or both.

[0052] A solid surface may comprise or consist of non-porous or porous particles, a porous membrane, a porous filter, or a porous monolith. In certain embodiments, a solid surface comprises or consist of particles. Particles may be spherical, roughly spherical or non- spherical, and may be of a size (average diameter) ranging from 10 μιη to 5 mm, 10 μιη to 1 mm, or 100 um to 500 nm. In certain embodiments, a solid surface comprises a matrix. A solid surface may further comprise or consist of compound constructions, for example in which particles are embedded in a reticular matrix, sandwiched between membranes, or both.

[0053] A binding agent can be covalently or non-covalently attached to a solid surface. In some embodiments, a binding agent is covalently attached to a solid surface. In certain embodiments, a binding agent is covalently attached to a bead or particle. A binding agent can be attached to a solid surface by a linker. Any suitable method can be used to attach a binding agent to a solid surface.

[0054] In certain embodiments, the membranes are in the form of porous-walled hollow fibers. In certain embodiments, the device contains a porous reticular arrangement of fibers, where such fibers are the substrates to which the first surface-bound ligand and an additional surface-bound ligand are attached.

[0055] In certain embodiments, a device described herein is configured for a

chromatography. In certain embodiments, a device described herein is configured for apheresis or plasma exchange. The portion of a device that contacts the blood or blood product obtained from a subject is often sterile and suitable for medical use. In some embodiments, a device comprises a cylinder, cartridge or column that contains a solid surface comprising a binding agent. In certain embodiments, device consists of a housing that contains the solid surface to which the binding agent is attached. In certain

embodiments, a device contains porous or non-porous particles sandwiched between porous membranes, or frits. In certain such embodiments, the particles are the solid surface to which binding agent is attached. In certain embodiments, a device contains porous or non-porous particles sandwiched between woven or amorphous fibrous filters. In certain such embodiments, the particles are the solid surface to which the binding agent is attached, and the fibrous filters are substantially inert. In certain embodiments, the device contains porous or non-porous particles sandwiched between woven or crystalline frits, and the frit is substantially inert. In certain embodiments, the device contains porous or non-porous particles embedded in a reticular polymer network. In certain such embodiments, particles are the solid surface to which binding agent is bound and the reticular polymer network is substantially inert. In certain embodiments, the device comprises particles and the particles are confined between membranes, monoliths, a reticular polymer network, woven or amorphous fiber filters, crystalline frits, or a combination thereof. In certain embodiments, a device contains one or more porous membranes and at least one of such membranes is a solid surface to which the binding agent is attached.

[0056] In certain embodiments, the chemical surface of one or more components of a device may be relatively inert or of such a relatively low surface area as to make no significant contribution to the chemical functionality of the device. In certain such embodiments, the chemical surface which is relatively inert or of relatively low surface area is configured so as to create structural integrity, or direct flow of liquids there through, or physically block, entrap, or entrain insoluble materials to prevent them from interfering with the effective use of the device.

[0057] In certain embodiments, a method described herein comprises extracorporeal depletion or removal of a transforming growth factor beta (TGFp) polypeptide, or complex thereof, from blood or a blood product obtained from a subject. A method may comprise, in certain embodiments, contacting the blood or blood product with a solid surface comprising a binding agent that specifically binds to a TGFp polypeptide. An antibody that specifically binds to a TGFp polypeptide will, upon contact with blood or a blood product that comprises a TGFp polypeptide, form a bound complex comprising the TGFp polypeptide and the binding agent, where the TGFp polypeptide is retained on the solid surface. The blood or blood product is then, in certain embodiments, separate from the solid surface. Where the solid surface is retained inside of a device, such as a column, cartridge or cylinder, the blood is separated from the solid surface by removing or expelling the blood or blood product from the device. In some embodiments, blood or a blood product is obtained directly from a subject, the blood or blood product then enters a device comprising a TGFp specific antibody, bound complexes are formed and retained on the solid surface, and the blood or blood product is returned to the subject, often by way of infusion.

[0058] In certain embodiments, a device comprises a plasma exchange system or an apheresis system. Plasma exchange systems are known in the art and are commercially available. Any suitable commercially available plasmapheresis or plasma exchange system that can separate the blood plasma fraction from the cellular fraction can be adapted for use in a method described herein. Non-limiting examples of suitable plasma exchange systems include TERUMO OPTIA and SPECTRA OPTIA system sold by TerumoBCT. In some embodiments, a suitable dialysis machine can be retrofitted with device described herein.

[0059] In some embodiments, a device described herein comprises an input port and an output port configured for continuous flow of blood or a blood product obtained from a subject through the device. Any suitable flow rate can be used. In some embodiments a flow rate of blood, or a blood product through a device described herein is 0.1 to 300 ml/min, or 1 to 100 ml/minute. In certain embodiments, blood or a blood product flows from the subject, into the input port where the blood or blood product is contacted with the solid surface comprised within the device, and then exits the device through the output port where the blood or blood product is returned to the subject.

[0060] In some embodiments, the disease or disorder is a cancer, tumor or metastasis. Non-limiting examples include hepatocellular cancer, mesothelioma; solid cancers or tumors, non-limiting examples of which include visceral tumors such as melanomas, breast, pancreatic, uterine and ovarian cancers, testicular cancer, including seminomas, gastric or colon cancer, hepatomas, adrenal, renal and bladder carcinomas, lung, head and neck cancers and brain tumors/cancers; carcinomas, non-limiting examples of which include respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, melanomas, neuroblastoma, squamous cell carcinoma of the head and neck, carcinomas of the uterus, cervix, lung, prostate, breast, head and neck, colon, pancreas, testes, adrenal, kidney, esophagus, stomach, liver and ovary; adenocarcinomas, sarcomas (e.g., lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma,

leiomyosarcoma, rhabdomyosarcoma and fibrosarcoma); neural neoplasias (e.g., glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma and oligodendrocytoma); Lymphomas, myelomas, and leukemias, non-limiting examples of which include acute and chronic lymphoblastic leukemia, myeloblastic leukemia, multiple myeloma, poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia), acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM), non-Hodgkin lymphoma and variants, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

EXAMPLES

Example 1

[0061 ] Demonstration of the removal of TGFpi was accomplished by fabricating a 1 imL column using Ultraflow-6 bead matrix format. An antibody to the latent TGFp complex, 7H4 was obtained from Biolegend, San Diego, CA. The amount of 5 mg of 7H4 antibody was coupled to the Ultraflow-6 beads. A solution of phosphate buffered saline and 0.05% BSA was spiked with Latent TGFp to 10 ng/mL This solution was run through the column at the sequential flow rates of 2,1 ,0.5,1 and 2 imL/min and fractions were collected. TGFp was measured using the MSD Mesoscale Discovery TGFp kit and the concentration of TGFp in the stock and flow through samples was measured. From this data the % of TGFp extracted was calculated and graphed in Figure 4. A control column consisting of bovine serum albumin coupled to Ultraflow-6 beads was used. The average amount of TGFp that was captured from the original stock solution was 34.8 ± 4.9% This was highly significant at the level of p = 0.0008 compared to control. It should be noted that the use of a column with an anti-TGFp antibody (1 D1 1 ) that binds to all three TGFp isoforms was unable to capture latent TGFp complexes. For this antibody solutions or plasma must be treated with low pH buffers to release TGFp from the complex in order to obtain binding.

Advantages of an Apheresis Approach

[0062] 1 . Control over therapeutic delivery. The monitoring of the patient responses during therapeutic administration is done in real time and can be discontinued at will. This is important safety consideration since removal of drug or biologies from the patient is precluded upon observations of toxicity.

[0063] 2. For drugs and biologies, parameters such as effective dose determinations, toxicity, elimination rate constants and so forth, must be determined as well as off target effects. Apheresis therapy is directed toward the simple removal of the target and is amenable to patient specific treatment in terms of frequency of administration and duration as required. For example, patients with a larger tumor burden may require a greater number of treatments.

[0064] 3. Apheresis is an on demand therapy that is independent of the parameters of drug or biologic administration since foreign substances are not introduced into the patient. This circumvents the potential for long term exposure to substances that can engender unwanted immune responses that may attenuate drug or biologic potency.

[0065] 4. Apheresis is transient and may be more effective as an acute immune-stimulatory model than long term drug or biologic delivery. It is known that perturbation of biologic systems may induce adverse compensatory feed-back responses which mitigate the potential for immune activation.

[0066] 5. Therapeutic targets can be quantified by measuring the amount of their removal whereas monitoring their inactivation by antibody therapy is difficult to quantify. With apheresis, this can be done by obtaining samples of pre- and post- column plasma during treatment and determination of the target protein concentrations. The total amount of target protein can be determined by elution of the columns that were used. In addition, changes in the blood composition of other potentially important markers can be followed over time post therapy.

[0067] Included in Example 2 are references to the generation and use of specific TGFpi antigens (26-31 ).

Example 2 - References

[0068] The following references are incorporated by reference in their entirety.

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22. Nam JS, Terabe M, Mamura M, Kang MJ, Chae H, Stuelten C, Kohn E, Tang B, Sabzevari H, Anver MR, Lawrence S, Danielpour D, Lonning S, Berzofsky JA, Wakefield LM. An anti-transforming growth factor beta antibody suppresses metastasis via cooperative effects on multiple cell compartments. Cancer Res. 2008;68(10):3835-43. doi:

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23. Loeys BL, Schwarze U, Holm T, Callewaert BL, Thomas GH, Pannu H, De Backer JF, Oswald GL, Symoens S, Manouvrier S, Roberts AE, Faravelli F, Greco MA, Pyeritz RE, Milewicz DM, Coucke PJ, Cameron DE, Braverman AC, Byers PH, De Paepe AM, Dietz HC. Aneurysm syndromes caused by mutations in the TGFp receptor. N Engl J Med.

2006;355(8):788-98. doi: 10.1056/NEJMoa055695. PubMed PMID: 16928994.

24. Lonning S, Mannick J, McPherson JM. Antibody targeting of TGFp in cancer patients. Curr Pharm Biotechnol. 201 1 ;12(12):2176-89. PubMed PMID: 21619535.

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26. Zou Z, Sun PD. Overexpression of human transforming growth factor-betal using a recombinant CHO cell expression system. Protein Expr Purif. 2004;37(2):265-72. doi:

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27. Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFpeta activation. J Cell Sci. 2003;1 16(Pt 2):217-24. PubMed PMID: 12482908. 28. Puthawala K, Hadjiangelis N, Jacoby SC, Bayongan E, Zhao Z, Yang Z, Devitt ML, Horan GS, Weinreb PH, Lukashev ME, Violette SM, Grant KS, Colarossi C, Formenti SC, Munger JS. Inhibition of integrin alpha(v)beta6, an activator of latent transforming growth factor-beta, prevents radiation-induced lung fibrosis. Am J Respir Crit Care Med.

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PMC2597394.

Example 3 - Sequences

[0069] TGFpl (Human) -(SEQ ID NO:1 )

[0070] Features:

Signal peptide - amino acids 1 -29

Latency-associated peptide - amino acids 30-278

Mature TGFpi Peptide- amino acids 279-390

MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLA SPPS QGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKF K QSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNR LL APSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDL ATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLG W KWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVY Y VGRKPKVEQLSNMIVRSCKCS

[0071 ] TGFp2 (Human) -(SEQ ID NO:2)

[0072] Features:

Signal peptide - amino acids 1 -20

Latency-associated peptide - amino acids 21 -302

Mature TGFp2 Peptide- amino acids 303-414

MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDY PEPEEV

PPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPP TFYRPY

FRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQR YIDSKV

VKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEEL EARFA

GIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAY CFRN

VQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNT INPE

ASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS

[0073] TGFp3 (Human) -(SEQ ID NO:3) [0074] Features:

Signal peptide - amino acids 1 -23

Latency-associated peptide - amino acids 24-300

Mature TGFP3 Peptide- amino acids 301 -412

MKMHLQRALVVLALLNFATVSLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPP EPTVMT

HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNEL AVCP

KGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIA KQRYIG

GKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHE VMEIKF

KGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYC FR

NLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYN TLN

PEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS