WO2020243228A1 - Antibodies to polyphosphate decrease fibrosis - Google Patents

Abstract

Polyphosphate activates the intrinsic pathway of coagulation that also induces inflammation. Hypercoagulable and hyperinflammatory challenge are mediators contributing to endothelial dysfunction, organ failure and death, which occur in many pathological conditions. Upon platelet activation, Polyphosphate is released into the microenvironment, where it has been shown to promote coagulation and act as a proinflammatory molecule. The inventors have found that platelet- sized polyphosphate promotes migration of fibroblasts, and also induces smooth muscle actin expression and the formation of stress fibers in fibroblasts, both of which are hallmarks of myofibroblast differentiation. Myofibroblasts play roles in driving both wound healing and fibrosis. This work suggests that polyphosphate not only promotes early wound healing through enhancing fibrin clot formation, but also may play a role in the later stages of wound healing and potentially the progression of fibrotic diseases by recruiting and inducing the differentiation of fibroblasts into myofibroblasts. As such, polyphosphates can be targeted by inhibitors for the treatment of aberrant/pathologic fibrosis, such as anti-polyphosphate antibodies and polyphosphatase enzymes, as well as used as biomarkers for diagnosis, prognosis, and treatment response indicators to provide guidance for treatment plans.

Description

ANTIBODIES TO POLYPHOSPHATE DECREASE FIBROSIS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under contract number UM1 HL120877 and R35 HL135823 awarded by the National Institutes of Health. The Government has certain rights in the invention. PRIORITY CLAIM

This application claims benefit of priority to U.S. Provisional Application Serial No. 62/853,326, filed May 28, 2019, the entire contents of which are hereby incorporated by reference. BACKGROUND

I. Field

The present disclosure relates to the field of medicine, disease and biology. In particular, it relates to the identification and targeting of polyphosphates (Poly-P) in fibrotic disease states. II. Related Art

Anionic polyphosphates (Poly-Ps) are linear polymers of orthophosphate units xxxxxd by high-energy phosphoanhydride bonds. The size of the chains varies from a few phosphates to thousands of phosphates. Particularly, bacterial polyphosphates have long chains and are stored in the inclusions called acidocalcisomes. Although polyphosphate has been identified in all cell types ranging from bacteria to humans, its role in higher eukaryotic organisms remains largely enigmatic. In microorganisms, Poly-P plays an important function as a store of phosphorus and energy, in cation homeostasis, and in adaptation to stress conditions. Eukaryotic Poly-Ps are shorter and are contained in platelet dense bodies, lysosomes, mitochondria and nuclei.

Recent evidence obtained over last decade demonstrated that Poly-Ps are potent inducers of intrinsic/contact pathway of coagulation and have important prothrombotic and proinflammatory effects. Poly-P is procoagulant because of its ability to: (i) enhance thrombin generation and fibrin-clot structure; (ii) accelerate activation of FXII, FXI and the contact (kallikrein-kinin) pathway; and (iii) abrogate the anticoagulant activity of tissue factor pathway of nuclear factor k-B (NFkB) and downstream induction of proinflammatory cytokines.

Poly-Ps are released in blood from bacteria during pathological conditions such as sepsis or from the damaged eukaryotic cells during sterile inflammation and thrombotic events. Platelets and other cells that contain Poly-P (e.g., mast cells) release Poly-P when are activated or when the content of mitochondria and lysosomes is released during cell death and during sepsis induced by bacterial species that contain Poly-P. Targeting Poly-P has the potential to attenuate inflammatory and thrombotic complications in a large spectrum of diseases.

In accordance with the disclosure, there is provided a method of inhibiting a fibrosis or a fibrotic condition involving extracellular polyphosphate toxicity in a subject comprising administering to the subject an anti-polyphosphate antibody or antigen-binding fragment thereof. The fibrotic condition may be fibrosis of lung, liver, heart, brain, artery, knee, shoulder, intestine, hands, fingers, kidney, uterus, vagina, gall bladder, bile duct, skin, mediastinum, bone marrow, penis, or soft tissue. The fibrotic condition may comprise pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced injury, such as radiation-induced lung injury, chemotherapy-induced fibrosis, cirrhosis of the liver, atrial fibrosis, endomyocardial fibrosis, old myocardial infarction, glial scar, arterial stiffness, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, adhesive capsulitis or trauma. The subject may be selected from a group consisting of human, dog, cat, horse, monkey, mouse, rat, rabbit, sheep, goat, cow, and pig.

The anti-polyphosphate antibody may be a monoclonal antibody or comprised in a polyclonal antiserum. The anti-polyphosphate antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab)₂ fragment, or Fv fragment. The anti-polyphosphate antibody may be a chimeric antibody or is bispecific antibody. The anti- polyphosphate antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

The method may further comprise administering to the subject a second anti-fibrosis therapy, such as an anti-histone antibody or a polyphosphate-degrading enzyme The method may further comprise assessing polyphosphate content in a serum or plasma sample from the subject before or after administering the antibody, such as assessing performed after administering the antibody, wherein the method further comprises adjusting a dosage of the administered anti-polyphosphate antibody responsive to the extracellular polyphosphate content. Assessing may comprise ELISA or Western blotting using anti-polyphosphate antibodies. condition involving extracellular polyphosphate toxicity in a subject comprising administering to the subject a polyphosphate-degrading enzyme. The fibrotic condition may be fibrosis of lung, liver, heart, brain, artery, knee, shoulder, intestine, hands, fingers, kidney, uterus, vagina, gall bladder, bile duct, skin, mediastinum, bone marrow, penis, or soft tissue. The fibrotic condition may comprise pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced injury, such as radiation-induced lung injury, chemotherapy-induced fibrosis, cirrhosis of the liver, atrial fibrosis, endomyocardial fibrosis, old myocardial infarction, glial scar, arterial stiffness, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, adhesive capsulitis or trauma.

The subject may be selected from a group consisting of human, dog, cat, horse, monkey, mouse, rat, rabbit, sheep, goat, cow, and pig. The polyphosphate-degrading enzyme may be bacterial exopolyphosphatase, such as PPX, or a yeast exopolyphosphatase, such as ScPPX1. The method may further comprise administering to the subject a second anti-fibrosis therapy, such as an anti-histone antibody. The method may further comprise assessing polyphosphate content in a serum or plasma sample from the subject before or after administering the antibody. Assessing may be performed after administering the antibody, and the method may further comprise adjusting a dosage of the administered anti-polyphosphate antibody responsive to the extracellular polyphosphate content. Assessing may be performed before administering the polyphosphate-degrading enzyme. Assessing may comprise ELISA or Western blotting using anti-polyphosphate antibodies.

The use of the word“a” or“an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.”

It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions and kits of the disclosure can be used to achieve methods of the disclosure.

Throughout this application, the term“about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.”

As used in this specification and claim(s), the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as “have” and“has”),“including” (and any form of including, such as“includes” and“include”) or“containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIG. 1. Representative structure of inorganic Poly-P, single unit within closed brackets.

FIG. 2. Time to clot differences in 5 different trials, 4 of which have Poly-P. Results demonstrate that with sufficient concentration of anti-Poly-P antibodies (e.g., PP2055) that Poly-P effect on time to clot is almost neutralized.

FIGS. 3A-C. Platelet-sized polyphosphate induces myofibroblast differentiation. (FIG.3A) Sub-confluent, cultured NIH-3T3 fibroblasts were incubated in DMEM medium containing 1.5% bovine calf serum for 48 hours in the absence (control) or presence of 5 mM chemically synthesized inorganic polyphosphate (Poly-P) of approximately the size secreted by activated platelets. Cells were subsequently fixed, permeabilized and stained using a monoclonal antibody against a-smooth muscle actin (a-SMA) and fluorescently labeled secondary antibody, then imaged using a confocal microscope. Typical fields are shown in FIG. 3A. Myofibroblasts are traditionally identified through an increase in a- SMA levels localized along actin stress fibers, as well as an enlarged cell body. (FIGS.3B & 3C) Fluorescent integrated density (the product of area and mean fluorescent value) was determined using ImageJ for cells treated for 48 hours with the indicated concentrations of polyphosphate. In FIG. 3B, total integrated a-SMA fluorescence is plotted versus the concentration of polyphosphate that the cells were exposed to. In FIG. 3C, the percent of cells positive for a stress-fiber pattern (defined as distinct staining localized actin fibrils as seen in the second image of FIG. 3A) of a-SMA fluorescence is plotted versus polyphosphate concentration. All values are mean ± SEM, n = 5, with a minimum of 25 cells analyzed for each individual experiment and condition. * indicates p < 0.05, ** p <0.01 compared to the no-polyphosphate control (paired t-tests).

FIGS. 4A-B. Platelet-sized polyphosphate increases collagen production. (FIGS. 4A & 4B) Sub-confluent NIH-3T3 fibroblasts were incubated in DMEM medium containing 1.5% bovine calf serum for 48 hours in the presence or absence of 1 mM platelet- sized polyphosphate (Poly-P) as performed in FIGS.3A-C. Cells were subsequently fixed, a fluorescently labeled secondary antibody, then imaged using a confocal microscope. In FIG.4A, fluorescent integrated density (as defined in FIGS.3A-C) was determined using ImageJ. Panel B shows typical fields. All values are mean ± SEM, n = 5 with a minimum of 25 cells analyzed for each individual experiment and condition. ** indicates p <0.01 compared to the no-polyphosphate control (paired t-tests).

FIGS.5A-B. Platelet releasates induce myofibroblast differentiation. (FIGS.5A & 5B) Releasates were generated by treating human platelets (at 1 x 10⁹/mL in Tyrode’s buffer) with Thrombin Receptor-Activating Peptide (TRAP). Platelets were removed by centrifugation, and the supernatant was collected (termed“platelet releasate”). The protein concentration in releasates was determined using a NanoDrop spectrophotometer, and the releasates were subsequently diluted in Tryode’s buffer to a protein concentration of 300 mg/ml. Some of the releasates were heated at 95 °C for 30 minutes to inactivate any protein activity, then cooled to room temperature (polyphosphate is heat-resistant, and many control experiments have shown that its activity is unaltered by this heat treatment). Some of the heated or non-heated releasates were subsequently treated with 40 mg/ml of a recombinant polyphosphate-degrading enzyme, yeast exo-polyphosphatase (ScPPX1), for 1 hour at 37 °C. The variously treated releasates, or Tyrode’s buffer control, were diluted twentyfold into DMEM containing 1.5% bovine calf serum and incubated with subconfluent NIH-3T3 fibroblasts for 48 hours. Cells were subsequently fixed, permeabilized and stained for a-SMA actin as done in FIGS. 3A-C and imaged using a confocal microscope. Fluorescent integrated density was determined using ImageJ, and cells were scored for a-SMA localized to actin stress fibers as done in FIGS. 3A-C. All values are mean ± SEM, n = 3 with a minimum of 25 cells analyzed for each individual experiment and condition. * indicates p < 0.05, ** p <0.01 compared to the no- polyphosphate control (unpaired t-tests).

FIGS. 6A-B. Poly-P antibody restores kidney morphology in rat renal ischemia- reperfusion model. Renal ischemia-reperfusion injury was performed by isolating the left kidney and using a microvascular clamp (bulldog) on the renal pedicle to restrict blood flow (both arterial and venous) (Dominguez et al., 2017). (FIG. 6A) MR image shows damage (such as necrosis, inflammation and early signs of fibrosis) to upper cortical and medullary regions (red arrow). Right kidney was the control kidney that was left alone. (FIG.6B) MR image of a Poly-P antibody-treated kidney (left), that underwent ischemia- to the control kidney on the right.

FIGS. 7A-D. Poly-P antibody restores kidney morphology in rat renal ischemia- reperfusion model. Histologically, Poly-P antibody therapy restores kidney tissue morphology to normal. Histological image shows damage to renal cells (white arrows; cells undergoing necrosis– irregular shapes and shrinking, and what may appear as early signs of fibrosis) in IRI kidney (FIG.7A). Right kidney is the control kidney that was left alone (FIG. 7B). Histological image of a Poly-P antibody-treated kidney (left), that underwent ischemia-reperfusion injury and was treated 30 min. prior to ischemia, appears normal (FIG. 7C), and similar to the control kidney on the right (FIG. 7D). Cells appear well- rounded, with no irregular cell membrane (as above), and no apparent necrosis. Magnification is 20x for all slides.

FIG. 8. Poly-P antibody restores kidney morphology in rat renal ischemia- reperfusion model. Quantitative data for MRI signal intensities (SI) indicates a significant decrease in MRI SI due to ischemia-reperfusion injury in the untreated kidney (relative to control kidney), and that Poly-P Ab treatment increases MRI SI to normal levels in the treated ischemic kidney (compared to the treated control kidney).

FIGS. 9A-D. Poly-P antibody restores kidney metabolites in rat renal ischemia- reperfusion model. (FIG. 9A) Renal MR Spectroscopy (MRS) was done by isolating regions in both left (red) (ischemic) and right (left) (control) kidneys. (FIG. 9B) Representative MR spectrum shows elevated TMA (trimethylamines) metabolites in ischemic kidney (left, red square, FIG.9A). TMA has been previously used as a marker of renal ischemia-reperfusion injury (Serkova, 2005). Reduced lipids are also observed, possibly indicating cell injury. (FIG.9C) Representative MR spectrum of a control kidney (right, yellow square region in FIG.9A). (FIG.9D) Representative MR spectrum of a Poly- P antibody-treated ischemic kidney. PC = phosphatidyl choline.

FIG. 10. Poly-P antibody restores kidney metabolites. Kidney metabolites TMA (trimethylamines) are significantly elevated in ischemia reperfusion injury (Serkova, 2005). Poly-P antibody treatment restores kidney TMA levels to near normal levels. Percent change in TMA levels was done by comparing ischemic kidney metabolite levels to control kidney metabolite levels.

FIGS.11A-B. Poly-P antibody restores kidney vascular perfusion rates to normal in rat renal ischemia-reperfusion model. Renal vascular perfusion rates were obtained by perfusion imaging method. (FIG.11A) Representative MR perfusion map shows decreased rTBF in untreated ischemic kidney (left, red square), compared to normal perfusion in control kidney (right, yellow square). (FIG. 11B) Representative MR perfusion map of a Poly-P antibody-treated ischemic kidney (left, red square) that appears to be similar to the control kidney (right, yellow square).

FIG. 12. Poly-P antibody restores kidney vascular perfusion to normal. Kidney perfusion rates are significantly reduced in ischemia reperfusion injury, both in the cortex and medulla regions. Poly-P antibody treatment restores kidney perfusion rates to near normal levels. Percent change in perfusion rates was done by comparing ischemic kidney perfusion rates to control kidney perfusion rates. Kidney perfusion rates are measured as mL/(100 g x min). ****p<0.0001.

FIG. 13. Polyphosphate enhances fibroblast migration. Sub-confluent NIH-3T3 fibroblasts were incubated either in“Serum Free” conditions (DMEM without bovine calf serum but supplemented with insulin-transferrin-selenium (ITS, Gibco, catalog # 41400- 045)) or in“1% Serum” conditions (DMEM with 1% bovine calf serum) in trans-well migration plates (ThermoFisher, catalog # 140629). Thus, NIH-3T3 cells were seeded in the top chamber of the trans-well migration plate using either serum-free medium or 1% serum-containing medium, and polyphosphate was added to the same medium in the bottom chamber, in order to generate a gradient of polyphosphate. After 24 hours, fibroblasts that had migrated to the bottom chamber (towards the gradient of polyphosphate) were collected and assayed using a fluorescent-based cell quantification assay (ThermoFisher, catalog # C7026) that measures the DNA content of cell lysates. Samples were normalized to the relative fluorescent units (RFU) of the no-polyphosphate control, which was assigned a value of 100%. All values are mean ± SEM, n = 5. For statistical significance, * indicates p < 0.05, while *** indicates p <0.005 (compared to the no-polyphosphate control, in paired t-tests).

As discussed above, Poly-P has been demonstrated to be involved in inflammatory signaling. The inventors hypothesized that Poly-P might also play a role in the development or progression of fibrotic diseases as a result of chronic inflammation, and the data reported here support that hypothesis. Moreover, inhibition of Poly-P should provide prophylaxis from or treatment of fibrotic disease states that are driven, at least in part, by Poly-P.

One candidate inhibitor of Poly-P would be an anti-Poly-P antibody. Initially, the ubiquitous nature of Poly-P raised a concern that it may not be possible for an animal to mount an immune response against this molecule, owing to immune tolerance. In addition, the simple, repeating structure of Poly-P raised concerns about the feasibility of generating an antibody as such simple molecules may not be antigenic. Finally, it was very unclear that antibodies to Poly-P, even if they could be generated, would be able to effectively block the procoagulant activity of Poly-P (see FIG. 1). However, the inventors have demonstrated the feasibility of generating blocking/neutralizing anti-Poly-P antibodies.

Another inhibitor is a polyphosphate-degrading enzyme, such as a polyphosphatase which catalyzes the hydrolysis of inorganic polyphosphate. Exopolyphosphatases begin at the ends of the polyphosphate chain and cleaves the phospho-anhydride bonds to release orthophosphate as it moves along the polyphosphate molecule.

Thus, there is provided a method of inhibiting a fibrotic condition involving extracellular Poly-P toxicity in a subject comprising administering to the subject an inhibitor of Poly-P toxicity, such as anti-Poly-P antibody or Poly-P-degrading enzyme, optionally with an anti-histone antibody. One may also measure a level of Poly-P in vivo for diagnosis, prognosis, and/or treatment response quantification. These and other aspects of the disclosure are described in greater detail below. I. Fibrosis and Fibrotic Disease States

Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. This can be a reactive, benign, or pathological state. In response to injury, this is called scarring, and if fibrosis arises from a single cell line, this is called a fibroma. Physiologically, fibrosis acts to deposit connective tissue, which can interfere with or totally inhibit the normal architecture and function of the underlying organ or tissue. Fibrosis can be used to describe the pathological state of excess deposition of fibrous tissue, as well as the process of connective tissue deposition in healing. Defined by the pathological of the affected tissue, it is in essence an exaggerated wound healing response which interferes with normal organ function.

Fibrosis is similar to the process of scarring, in that both involve stimulated fibroblasts laying down connective tissue, including collagen and glycosaminoglycans. The process is initiated when immune cells such as macrophages release soluble factors that stimulate fibroblasts. The most well characterized pro-fibrotic mediator is TGF beta, which is released by macrophages as well as any damaged tissue between surfaces called interstitium. Other soluble mediators of fibrosis include CTGF, platelet-derived growth factor (PDGF), and interleukin 4 (IL-4). These initiate signal transduction pathways such as the AKT/mTOR and SMAD pathways that ultimately lead to the proliferation and activation of fibroblasts, which deposit extracellular matrix into the surrounding connective tissue. This process of tissue repair is a complex one, with tight regulation of extracellular matrix (ECM) synthesis and degradation ensuring maintenance of normal tissue architecture. However, the entire process, although necessary, can lead to a progressive irreversible fibrotic response if tissue injury is severe or repetitive, or if the wound healing response itself becomes deregulated.

Fibrosis can occur in many tissues within the body, typically as a result of inflammation or damage, and examples include lungs (pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, or radiation-induced lung injury), liver (cirrhosis), heart (atrial fibrosis, endomyocardial fibrosis, old myocardial infarction), brain (glial scar) or others including arterial stiffness, arthrofibrosis (knee, shoulder, other joints), Crohn's disease (intestine), Dupuytren's contracture (hands, fingers), keloid (skin), mediastinal fibrosis (soft tissue of the mediastinum), myelofibrosis (bone marrow), Peyronie's disease (penis), nephrogenic systemic fibrosis (skin), progressive massive fibrosis (lungs), retroperitoneal fibrosis (soft tissue of the retroperitoneum), scleroderma/systemic sclerosis (skin, lungs), and some forms of adhesive capsulitis (shoulder). The present disclosure contemplates intervening in any of these fibrotic disease states that involve the release of Poly-Ps. II. Polyphosphates

In biology, inorganic Poly-Ps are found in many organs of subjects, including humans. Poly-P is released from dense granules in platelets and from certain secretory granules in mast cells. Poly-P is accumulated by many infectious microorganisms, and Poly-P can be critical for cell viability in bacteria. Damaged microorganisms may release Poly-P, and the released Poly- long) are considerably shorter than the Poly-P found in infectious microorganisms (ranging up to hundreds of phosphate units long, and frequently in excess of 1000 phosphate units long). Bacterial Poly-P is very potent at triggering the contact pathway of blood clotting. Short Poly- P polymers are effective at accelerating certain blood clotting reactions in the common pathway of the plasma clotting cascade. Poly-P activates the intrinsic pathway of coagulation that also induces inflammation.

It is contemplated in the present disclosure that anti-Poly-P antibodies may block Poly- P toxicity and its action in driving fibrosis. The present disclosure also may employ antibodies that comprise modified, non-natural and/or unusual amino acids. The treatment may occur in vivo and can occur extracorporeally. For example, extracellular polyphosphate might be “cleaned” from or neutralized within blood flow through a cardiac bypass machine or extracorporeal life-support (e.g., PLS system from Maquet Getinge Group), by way of example and not limitation. III. Antibodies

It will be understood that polyclonal or monoclonal antibodies that bind immunologically to Poly-Ps will have use in several applications. These include diagnostic kits and methods of detecting Poly-Ps, as well as therapeutic intervention. Means for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, 1988; incorporated herein by reference). The term“antibody” as used herein is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab´, Fab, F(ab´)₂, single domain antibodies (DAB’s), Fv, scFv (single-chain Fv), and the like. A. Polyclonal Antisera

Polyclonal antisera are generally prepared by immunizing an animal with an immunogenic composition. In accordance with the present disclosure, the advantageous use of aged autoimmune mice eliminates the need for immunization as mice with this profile generate antibodies against autoantigens without any further treatment. An exemplary method using aged autoimmune mice is described below in the examples.

Antibody purification involves selective enrichment or specific isolation of antibodies from serum (polyclonal antibodies), ascites fluid or cell culture supernatant of a hybridoma cell crude to highly specific and can be classified as follows: Physicochemical fractionation: This involves process like differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on size, charge or other shared chemical characteristics of antibodies in typical samples, which isolates a subset of sample proteins that includes the immunoglobulins.

Class-specific affinity: This involves solid-phase binding of particular antibody classes (e.g., IgG) by immobilized biological ligands (proteins, lectins, etc.) that have specific affinity to immunoglobulins, which purifies all antibodies of the target class without regard to antigen specificity.

Antigen-specific affinity: This involves affinity purification of only those antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domains, which purifies all antibodies that bind the antigen without regard to antibody class or isotype. Unlike antibodies that are developed as monoclonal antibody hybridoma cell lines and produced as ascites fluid or cell culture supernatant, where the target antibody is (for most practical purposes) the only immunoglobulin in the production sample, polyclonal antibodies (serum samples) typically employ antigen-specific affinity purification is required to prevent co-purification of nonspecific immunoglobulins.

For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The procured blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody or a peptide bound to a solid matrix or protein A followed by antigen (peptide) affinity column for purification. B. Monoclonal Antibodies

mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No.4,196,265, incorporated herein by reference. As described above, i.e., relies on the animal’s ability to produce antibodies against self-antigens.

Somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens or lymph nodes. Spleen cells and lymph node cells may be used, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage. The spleen of animal is removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp.65-66, 1986; Campbell, pp.75-83, 1984; each incorporated herein by reference).

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies, about 1×10^-6 to 1×10^-8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. are selected. Typically, selection of hybridomas is performed by culturing the cells by single- clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, toxicity assays, plaque assays, dot immunobinding assays, and the like. The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways.

A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.

The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used to generate monoclonals. For this, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells e.g., normal-versus-tumor cells. The advantages of this approach over conventional hybridoma techniques are that approximately 10⁴ times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies. In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross-reactivity or diminished off-target binding. Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document. The following is a general discussion of relevant goals techniques for antibody engineering.

Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.

Recombinant full-length IgG antibodies can be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 (e.g., Freestyle) cells or CHO cells, and antibodies can be collected and purified from the 293 or CHO cell supernatant. Other appropriate host cells systems include bacteria, such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without engineering for human-like glycans), algae, or in a variety of non-human transgenic contexts, such as mice, rats, goats or cows.

Expression of nucleic acids encoding antibodies, both for the purpose of subsequent antibody purification, and for immunization of a host, is also contemplated. Antibody coding sequences can be RNA, such as native RNA or modified RNA. Modified RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1-methyl-pseudouridine (N1mY) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. In addition to turning off the immune/eIF2a phosphorylation-dependent inhibition of translation, incorporated N1mY nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density on the mRNA. Increased ribosome loading of modified mRNAs renders them more permissive for initiation by favoring either ribosome recycling on the same mRNA or de novo vivo following inoculation with RNA. The RNA, whether native or modified, may be delivered as naked RNA or in a delivery vehicle, such as a lipid nanoparticle.

Alternatively, DNA encoding the antibody may be employed for the same purposes. The DNA is included in an expression cassette comprising a promoter active in the host cell for which it is designed. The expression cassette is advantageously included in a replicable vector, such as a conventional plasmid or minivector. Vectors include viral vectors, such as poxviruses, adenoviruses, herpesviruses, adeno-associated viruses, and lentiviruses are contemplated. Replicons encoding antibody genes such as alphavirus replicons based on VEE virus or Sindbis virus are also contemplated. Delivery of such vectors can be performed by needle through intramuscular, subcutaneous, or intradermal routes, or by transcutaneous electroporation when in vivo expression is desired.

The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g/L was achieved within 9 weeks of transfection.

Antibody molecules will comprise fragments (such as F(ab ), F(ab )₂) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. F(ab ) antibody derivatives are monovalent, while F(ab )₂ antibody derivatives are bivalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form “chimeric” binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.

In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (- 3.4), phenylalanine (-2.5), and tyrosine (-2.3).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG₁ can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency. with one or more further amino acid modifications that alter C1q binding and/or the complement dependent cytotoxicity (CDC) function of the Fc region of an IL-23p19 binding molecule. The binding polypeptide of particular interest may be one that binds to C1q and displays complement dependent cytotoxicity. Polypeptides with pre-existing C1q binding activity, optionally further having the ability to mediate CDC may be modified such that one or both of these activities are enhanced. Amino acid modifications that alter C1q and/or modify its complement dependent cytotoxicity function are described, for example, in WO/0042072, which is hereby incorporated by reference.

One can design an Fc region of an antibody with altered effector function, e.g., by modifying C1q binding and/or FcgR binding and thereby changing CDC activity and/or ADCC activity.“Effector functions” are responsible for activating or diminishing a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

For example, one can generate a variant Fc region of an antibody with improved C1q binding and improved FcgRIII binding (e.g., having both improved ADCC activity and improved CDC activity). Alternatively, if it is desired that effector function be reduced or ablated, a variant Fc region can be engineered with reduced CDC activity and/or reduced ADCC activity. In other embodiments, only one of these activities may be increased, and, optionally, also the other activity reduced (e.g., to generate an Fc region variant with improved ADCC activity, but reduced CDC activity and vice versa).

FcRn binding. Fc mutations can also be introduced and engineered to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc variants with improved binding to the FcRn have been described (Shields et al., (2001). High resolution mapping of the binding site on human IgG1 for FcgRI, FcgRII, FcgRIII, and FcRn and design of IgG1 variants with improved binding to the FcgR, (J. Biol. Chem.276:6591-6604). A number of methods are known that can result in increased half- life (Kuo and Aveson, (2011)), including amino acid modifications may be generated through techniques including alanine scanning mutagenesis, random mutagenesis and screening to Computational strategies followed by mutagenesis may also be used to select one of amino acid mutations to mutate.

The present disclosure therefore provides a variant of an antigen binding protein with optimized binding to FcRn. In a particular embodiment, the variant of an antigen binding protein comprises at least one amino acid modification in the Fc region of the antigen binding protein, wherein the modification is selected from the group consisting of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447 of the Fc region as compared to the parent polypeptide, wherein the numbering of the amino acids in the Fc region is that of the EU index in Kabat. In a further aspect of the disclosure the modifications are M252Y/S254T/T256E.

Additionally, various publications describe methods for obtaining physiologically active molecules whose half-lives are modified, see for example Kontermann (2009) either by introducing an FcRn-binding polypeptide into the molecules or by fusing the molecules with antibodies whose FcRn-binding affinities are preserved but affinities for other Fc receptors have been greatly reduced or fusing with FcRn binding domains of antibodies.

Derivatized antibodies may be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, particularly a human. Such alterations may result in a half- life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half- lives of the antibodies of the present disclosure or fragments thereof in a mammal, preferably a human, results in a higher serum titer of the antibodies or antibody fragments in the mammal, and thus reduces the frequency of the administration of the antibodies or antibody fragments and/or reduces the concentration of the antibodies or antibody fragments to be administered. Antibodies or fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or and the FcRn receptor.

Beltramello et al. (2010) previously reported the modification of neutralizing mAbs, due to their tendency to enhance dengue virus infection, by generating in which leucine residues at positions 1.3 and 1.2 of CH2 domain (according to the IMGT unique numbering for C-domain) were substituted with alanine residues. This modification, also known as“LALA” mutation, abolishes antibody binding to FcgRI, FcgRII and FcgRIIIa, as described by Hessell et al. (2007). The variant and unmodified recombinant mAbs were compared for their capacity to neutralize and enhance infection by the four dengue virus serotypes. LALA variants retained the same neutralizing activity as unmodified mAb but were completely devoid of enhancing activity. LALA mutations of this nature are therefore contemplated in the context of the presently disclosed antibodies.

Altered Glycosylation. A particular embodiment of the present disclosure is an isolated monoclonal antibody, or antigen binding fragment thereof, containing a substantially homogeneous glycan without sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, both of which may be attached to heavy chain or light chain constant regions respectively. The aforementioned substantially homogeneous glycan may be covalently attached to the heavy chain constant region.

Another embodiment of the present disclosure comprises a mAb with a novel Fc glycosylation pattern. The isolated monoclonal antibody, or antigen binding fragment thereof, is present in a substantially homogenous composition represented by the GNGN or G1/G2 glycoform. Fc glycosylation plays a significant role in anti-viral and anti-cancer properties of therapeutic mAbs. The disclosure is in line with a recent study that shows increased anti- lentivirus cell-mediated viral inhibition of a fucose free anti-HIV mAb in vitro. This embodiment of the present disclosure with homogenous glycans lacking a core fucose, showed increased protection against specific viruses by a factor greater than two-fold. Elimination of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells but appears to have the opposite effect on the ADCC activity of polymorphonuclear cells (PMNs).

The isolated monoclonal antibody, or antigen binding fragment thereof, comprising a substantially homogenous composition represented by the GNGN or G1/G2 glycoform exhibits increased binding affinity for Fc gamma RI and Fc gamma RIII compared to the same antibody GNGNF or GNGNFX containing glycoforms. In one embodiment of the present disclosure, the antibody dissociates from Fc gamma RI with a Kd of 1 x 10^-8 M or less and from Fc gamma RIII with a Kd of 1 x 10^-7 M or less.

Glycosylation of an Fc region is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O- linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequences are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Thus, the presence of either of these peptide sequences in a polypeptide creates a potential glycosylation site.

The glycosylation pattern may be altered, for example, by deleting one or more glycosylation site(s) found in the polypeptide, and/or adding one or more glycosylation site(s) that are not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). An exemplary glycosylation variant has an amino acid substitution of residue Asn 297 of the heavy chain. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original polypeptide (for O-linked glycosylation sites). Additionally, a change of Asn 297 to Ala can remove one of the glycosylation sites.

In certain embodiments, the antibody is expressed in cells that express beta (1,4)-N- acetylglucosaminyltransferase III (GnT III), such that GnT III adds GlcNAc to the IL-23p19 antibody. Methods for producing antibodies in such a fashion are provided in WO/9954342, WO/03011878, patent publication 20030003097A1, and Umana et al., Nature Biotechnology, 17:176-180, February 1999. Cell lines can be altered to enhance or reduce or eliminate certain post-translational modifications, such as glycosylation, using genome editing technology such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). For example, CRISPR technology can be used to eliminate genes encoding glycosylating enzymes in 293 or CHO cells used to express recombinant monoclonal antibodies.

Elimination of monoclonal antibody protein sequence liabilities. It is possible to engineer the antibody variable gene sequences obtained from human B cells to enhance their searching for sequence motifs associated with sites containing:

1) Unpaired Cys residues,

2) N-linked glycosylation,

3) Asn deamidation,

4) Asp isomerization,

5) SYE truncation,

6) Met oxidation,

7) Trp oxidation,

8) N-terminal glutamate,

9) Integrin binding,

10) CD11c/CD18 binding, or

11) Fragmentation

Such motifs can be eliminated by altering the synthetic gene for the cDNA encoding recombinant antibodies.

Protein engineering efforts in the field of development of therapeutic antibodies clearly reveal that certain sequences or residues are associated with solubility differences (Fernandez- Escamilla et al., Nature Biotech., 22 (10), 1302-1306, 2004; Chennamsetty et al., PNAS, 106 (29), 11937-11942, 2009; Voynov et al., Biocon. Chem., 21 (2), 385-392, 2010) Evidence from solubility-altering mutations in the literature indicate that some hydrophilic residues such as aspartic acid, glutamic acid, and serine contribute significantly more favorably to protein solubility than other hydrophilic residues, such as asparagine, glutamine, threonine, lysine, and arginine.

Stability. Antibodies can be engineered for enhanced biophysical properties. One can use elevated temperature to unfold antibodies to determine relative stability, using average apparent melting temperatures. Differential Scanning Calorimetry (DSC) measures the heat capacity, Cp, of a molecule (the heat required to warm it, per degree) as a function of temperature. One can use DSC to study the thermal stability of antibodies. DSC data for mAbs is particularly interesting because it sometimes resolves the unfolding of individual domains within the mAb structure, producing up to three peaks in the thermogram (from unfolding of the Fab, CH2, and CH3 domains). Typically unfolding of the Fab domain produces the strongest peak. The DSC profiles and relative stability of the Fc portion show characteristic differences for the human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. temperature using circular dichroism (CD), performed with a CD spectrometer. Far-UV CD spectra will be measured for antibodies in the range of 200 to 260 nm at increments of 0.5 nm. The final spectra can be determined as averages of 20 accumulations. Residue ellipticity values can be calculated after background subtraction. Thermal unfolding of antibodies (0.1 mg/mL) can be monitored at 235 nm from 25-95 °C and a heating rate of 1 °C/min. One can use dynamic light scattering (DLS) to assess for propensity for aggregation. DLS is used to characterize size of various particles including proteins. If the system is not disperse in size, the mean effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of surface structures, and particle concentration. Since DLS essentially measures fluctuations in scattered light intensity due to particles, the diffusion coefficient of the particles can be determined. DLS software in commercial DLA instruments displays the particle population at different diameters. Stability studies can be done conveniently using DLS. DLS measurements of a sample can show whether the particles aggregate over time or with temperature variation by determining whether the hydrodynamic radius of the particle increases. If particles aggregate, one can see a larger population of particles with a larger radius. Stability depending on temperature can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include proven methodologies for determining features of antibody stability. One can use an iCE approach to resolve antibody protein charge variants due to deamidation, C-terminal lysines, sialylation, oxidation, glycosylation, and any other change to the protein that can result in a change in pI of the protein. Each of the expressed antibody proteins can be evaluated by high throughput, free solution isoelectric focusing (IEF) in a capillary column (cIEF), using a Protein Simple Maurice instrument. Whole-column UV absorption detection can be performed every 30 seconds for real time monitoring of molecules focusing at the isoelectric points (pIs). This approach combines the high resolution of traditional gel IEF with the advantages of quantitation and automation found in column-based separations while eliminating the need for a mobilization step. The technique yields reproducible, quantitative analysis of identity, purity, and heterogeneity profiles for the expressed antibodies. The results identify charge heterogeneity and molecular sizing on the antibodies, with both absorbance and native fluorescence detection modes and with sensitivity of detection down to 0.7 µg/mL.

Solubility. One can determine the intrinsic solubility score of antibody sequences. The intrinsic solubility scores can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol HCDR3 of each antibody fragment such as a scFv can be evaluated via the online program to calculate the solubility scores. One also can determine solubility using laboratory techniques. Various techniques exist, including addition of lyophilized protein to a solution until the solution becomes saturated and the solubility limit is reached, or concentration by ultrafiltration in a microconcentrator with a suitable molecular weight cut-off. The most straightforward method is induction of amorphous precipitation, which measures protein solubility using a method involving protein precipitation using ammonium sulfate (Trevino et al., J Mol Biol, 366: 449-460, 2007). Ammonium sulfate precipitation gives quick and accurate information on relative solubility values. Ammonium sulfate precipitation produces precipitated solutions with well-defined aqueous and solid phases and requires relatively small amounts of protein. Solubility measurements performed using induction of amorphous precipitation by ammonium sulfate also can be done easily at different pH values. Protein solubility is highly pH dependent, and pH is considered the most important extrinsic factor that affects solubility.

Autoreactivity. Generally, it is thought that autoreactive clones should be eliminated during ontogeny by negative selection, however it has become clear that many human naturally occurring antibodies with autoreactive properties persist in adult mature repertoires, and the autoreactivity may enhance the antiviral function of many antibodies to pathogens. It has been noted that HCDR3 loops in antibodies during early B cell development are often rich in positive charge and exhibit autoreactive patterns (Wardemann et al., Science 301, 1374-1377, 2003). One can test a given antibody for autoreactivity by assessing the level of binding to human origin cells in microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometric cell surface staining (using suspension Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity also can be surveyed using assessment of binding to tissues in tissue arrays.

Preferred residues (“Human Likeness”). B cell repertoire deep sequencing of human B cells from blood donors is being performed on a wide scale in many recent studies. Sequence information about a significant portion of the human antibody repertoire facilitates statistical assessment of antibody sequence features common in healthy humans. With knowledge about the antibody sequence features in a human recombined antibody variable gene reference database, the position specific degree of“Human Likeness” (HL) of an antibody sequence can be estimated. HL has been shown to be useful for the development of antibodies in clinical use, like therapeutic antibodies or antibodies as vaccines. The goal is to increase the human likeness lead to significantly decreased efficacy of the antibody drug or can induce serious health implications. One can assess antibody characteristics of the combined antibody repertoire of three healthy human blood donors of about 400 million sequences in total and created a novel “relative Human Likeness” (rHL) score that focuses on the hypervariable region of the antibody. The rHL score allows one to easily distinguish between human (positive score) and non-human sequences (negative score). Antibodies can be engineered to eliminate residues that are not common in human repertoires. D. Single Chain Antibodies

A single chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma or B cell. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.

Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single- chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 × 10⁶ different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the V_H C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers. moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.

In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).

Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stabilizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero- bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).

It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is“sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, agent to the target site.

The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3 -dithiopropionate. The N- hydroxysuccinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

U.S. Patent 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.

U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques. E. Multispecific Antibodies

In certain embodiments, antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a binding that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcgR), such as FcgRI (CD64), FcgRII (CD32) and Fc gamma RIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies may also be used to localize cytotoxic agents to infected cells. These antibodies possess a pathogen-binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab )2 bispecific antibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-Fc gamma RIII antibody and U.S. Patent 5,837,234 discloses a bispecific anti-ErbB2/anti-Fc gamma RI antibody. A bispecific anti-ErbB2/Fc alpha antibody is shown in WO98/02463. U.S. Patent 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C_H2, and C_H3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two effect on the yield of the desired chain combination.

In a particular embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Patent 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the C_H3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Techniques exist that facilitate the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a humanized bispecific antibody F(ab')₂ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described (Merchant et al., Nat. Biotechnol.16, 677–681 (1998). doi:10.1038/nbt0798-677pmid:9661204). For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547-1553, 1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a V_H connected to a V_L by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

In a particular embodiment, a bispecific or multispecific antibody may be formed as a DOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Patents 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section of each of which is incorporated herein by reference.) Generally, the technique takes advantage of the specific and high-affinity binding interactions that occur between a dimerization and docking domain (DDD) sequence (AD) sequence derived from any of a variety of AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264; Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein, peptide or other molecule. Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences.

Charge modifications are particularly useful in the context of a multispecific antibody, where amino acid substitutions in Fab molecules result in reducing the mispairing of light chains with non-matching heavy chains (Bence-Jones-type side products), which can occur in the production of Fab-based bi-/multispecific antigen binding molecules with a VH/VL exchange in one (or more, in case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT publication no. WO 2015/150447, particularly the examples therein, incorporated herein by reference in its entirety). IV. Polyphosphate-Degrading Enzymes

There are a variety of polyphosphate degrading enzymes that are known the art. Any of these enzymes may be employed in the methods of this disclosure to inhibit fibrosis and/or treat fibrotic disease.

One polyphosphatase is exopolyphosphatase (PPX), a phosphatase enzyme that catalyzes the hydrolysis of inorganic polyphosphate. PPX is a processive exopolyphosphatase, which means that it begins at the ends of the polyphosphate chain and cleaves the phospho- anhydride bonds to release orthophosphate as it moves along the polyphosphate molecule. PPX has several characteristics which distinguish it from other known polyphosphatases, namely that it does not act on ATP, has a strong preference for long chain polyphosphate, and has a very low affinity for polyphosphate molecules with less than 15 phosphate monomers.

PPX plays an important role in the metabolism of phosphate and energy in all living organisms. It is especially important for maintenance of appropriate levels of intracellular polyphosphate, which has been implicated in a variety of cellular functions including response to stressors such as deficiencies in amino acids, orthophosphate, or nitrogen, changes in pH, nutrient downshift, and high salt, and as an inorganic molecular chaperone. phosphoesterase family. Both subfamilies within this super family share four N-terminus motifs but have different C-terminus moieties.

PPX activity is quantified by measuring the loss of radioactively labeled ³²P polyphosphate. PPX is mixed with a known quantity of labeled polyphosphate, and the hydrolysis reaction is stopped with perchloric acid (HClO4). The amount of remaining labeled polyphosphate is then measured by liquid scintillation counting.

PPX was discovered in 1993 and is part of the polyphosphate operon along with polyphosphate kinase, the enzyme which synthesizes polyphosphate. The Kornberg lab was very interested in polyphosphate and published a series of papers elucidating the metabolism and roles of polyphosphate in vivo. Their interest in polyphosphate led them to identify and characterize the polyphosphate operon (which includes polyphosphate kinase (PPK) and PPX) and develop a wide variety of assays and techniques for quantification of polyphosphate production and degradation, in vitro and in vivo. The results of these studies of polyphosphate by the Kornberg lab led Kornberg to speculate that due to its high energy and phosphate content and the degree to which it is conserved across species, polyphosphate may have been the precursor to RNA, DNA, and proteins.

The structure of PPX is characterized by the actin-like ATPase domain that is a part of this superfamily. In Aquifex aeolicus it contains a ribonuclease H-like motif that is made up of a five-stranded ß-sheet with the second strand antiparallel to the rest. A few of the strands are connected by helical segments that are longer in the C-terminal domain than in the N-terminal domain. Five alpha-helices are located in the C-terminal domain and only two are located in the N-terminal domain. The closed configuration of the enzyme is referred to as the type I structure. This configuration shares similar features to other members of this superfamily, including the N-terminal and C-terminal domains being separated by two alpha-helices centered on the structure. The more open arrangement of the domains displays rotational movement of the two domains around a single hinge region. The structural flexibility has been described as a "butterfly like" cleft opening around the active site.

In E. coli, exopolyphosphatase exists as a dimer, with each monomer consisting of four domains. The first two domains consist of three beta-sheets followed by an alpha-beta-alpha- beta-alpha fold. This is different from the previously described Aquifex aeolicus homolog which lacks the third and fourth domains. To date, 4 structures have been solved for this class of enzymes, with Protein Data Bank accession codes 1T6C, 1T6D, 1U6Z, and 2FLO. In E. coli, this region contains a loop between strands beta-1 and beta-2 with the amino acids glutamate and aspartate (E121, D143, and E150). These residues, along with K197 are critical for phosphate binding and ion binding which is commonly seen among other ASKHA (acetate and sugar kinases, Hsp70, actin). In A. aeolicus, the active site of the enzyme exists in a cleft between the two domains. It is seen that catalytic carboxyl groups in this cleft are important for the enzyme activity, specifically Asp141 and Glu148. The preference of exopolyphosphatase to bind to polyphosphate and not ATP has been contributed to the clashing that would occur between the ribose and adenosine of ATP and the side chains of N21, C169, and R267.

Exopolyphosphatase cleaves a terminal phosphate off of polyphosphate through the amino acid side chains of glutamate and lysine. Glutamate activates water, allowing it to act as a nucleophile and attack the terminal phosphate. The oxygen that was previously bridging the two phosphate atoms then abstracts a hydrogen from the nearby lysine residue.

Polyphosphates are utilized by exopolyphosphatase enzymes, which cleave portions of the chain of phosphates. These proteins play an essential role in the metabolism and maintenance of polyphosphates. Polyphosphate is located throughout the cytosol of each cell and is also present in the cell's organelles. There are many classes of exopolyphosphatases, each with their own unique localization and properties. It has been speculated that once the polyphosphates are broken down, they are involved with signaling molecules acting as secondary messengers. In E. coli, the regulation of polyphosphate metabolism is poorly understood.

Polyphosphate is a linear chain of phosphates linked together by phosphoanhydride bonds. Polyphosphate is found in all living organisms and plays an essential role in the organism’s survival. In bacteria, polyphosphate is used to store energy to replace adenosine triphosphate. It has also been shown to be involved with cell membrane formation and function, enzyme regulation, and gene transcriptional control. In mammals, polyphosphates are involved with blood coagulation and inflammation, immune response, bone tissue development, and brain function.

It has been shown in a yeast model that mutant yeast deficient in exopolyphosphatase activity had problems in respiration functions and metabolism of inorganic polyphosphates. Conversely, yeast strains that have higher levels exopolyphosphatase enzyme are shown to have no obvious growth defects under phosphate deficiency or excess phosphate conditions, of enzymes breaking the polyphosphate chains down.

Another possible polyphosphatase for use in accordance with the present disclosure is an endopolyphosphatase, an enzyme that catalyzes the chemical reaction

polyphosphate + n H₂O (n⁺1) oligophosphate

Thus, the two substrates of this enzyme are polyphosphate and H2O, whereas its product is oligophosphate. This enzyme belongs to the family of hydrolases, specifically those acting on acid anhydrides in phosphorus-containing anhydrides. The systematic name of this enzyme class is polyphosphate polyphosphohydrolase. Other names in common use include polyphosphate depolymerase, metaphosphatase, polyphosphatase, and polymetaphosphatase. V. Therapy

A. Treatments

In certain aspects, the present disclosure relates to the treatment of disorders that have as a component the production of extracellular Poly-Ps that results in fibrosis. By using agents that bind and block the function of the extracellular Poly-Ps, the inventors seek to reduce and inhibit the fibrosis driven by these molecules.

The present disclosure contemplates the use of inhibitors of extracellular Poly-P to treat a variety of disease states as specified above. The inventors contemplate the use of antibodies to Poly-Ps, as well as polyphosphate-degrading enzymes. Also contemplated are mixtures of agents, including at least one anti-Poly-P antibody and/or polyphosphate-degrading enzyme with at least one anti-histone antibody.

The therapies may include mAbs where at least one anti-Poly-P antibody is humanized by any suitable method, as are known to those of skill in the art, for therapeutic administration to humans. The therapies may include mAbs where at least one anti-Poly-P antibody is not humanized for therapeutic administration to humans. The therapies may include a polyphosphate-degrading enzyme that is an exopolyphosphatase.

Treatment regimens will vary depending on the severity and type of disease, the overall health and age of the patient, and various other conditions to be taken into account by the treating physician. Multiple doses or treatments may be applied, as well as“continuous” therapy where a small amount of the therapeutic agent is provided continually over an extended formulated to provide delayed, timed or extended release of the active form.

In addition, combinations of an inhibitor Poly-P with other treatments may be used by administration of a single composition or pharmacological formulation that includes both multiple agents, or by administering two distinct compositions or formulations, at the same time. Alternatively, one treatment may precede or follow administration of the other by intervals ranging from minutes to weeks. In embodiments where the two agents are applied separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that both agents would still be able to exert an advantageously combined effect. In such instances, it is contemplated that one would typically administer both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours may be used. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. It also is conceivable that more than one administration of a drug will be desired.

By way of illustration, the following permutations based on 3 and 4 total administrations are exemplary, where A represents a first inhibitor of Poly-P and B represents a second drug (including a second inhibitor of Poly-P or an anti-histone antibody): A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B Other combinations are likewise contemplated. B. Pharmaceutical Formulations and Routes of Administration

Where clinical applications are contemplated, pharmaceutical compositions anti-Poly- P antibodies, polyphosphate-degrading enzymes, and mixtures thereof, will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers to permit for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a vector or cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the agents of the therapeutic compositions.

The active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.

The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions of the present disclosure generally may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intraarterial, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be fluid injected at the proposed site of infusion, (see for example,“Remington’s Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards. VI. Examples

The following example is included to demonstrate embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1

NIH-3T3 cell culture and staining. Sub-confluent NIH-3T3 cells were seeded on glass coverslips in 24-well tissue culture plates at a density of 0.4 x 10⁴ cells / cm² in Dulbecco`s Modified Eagle Media (DMEM) containing 2.5% bovine calf serum and allowed to adhere overnight. The next day cells were stimulated with polyphosphate or platelet releasates in fresh DMEM containing 1.5% serum and allowed to incubate for 48 hours. Cells were washed twice with TBS (Tris-buffered saline) then fixed using 4% paraformaldehyde for 20 minutes. With three washes in TBS for 5 minutes between each step, cells were permeabilized in 0.2% triton X-100 for 10 minutes, blocked in TBST (Tris-buffered saline + 0.05% Tween-20) + 5% bovine serum albumin (BSA) for 1 hour, incubated with primary antibody in TBST + 1% BSA for 1 hour, incubated with secondary antibody in TBST + 1% BSA for 1 hour, then mounted onto glass slides using antifade mounting medium (ProLong Gold from ThermoFisher, catalog number P36930). Anti-alpha-smooth muscle actin antibody (ThermoFisher, catalog number 14976082) was used at a 1:1000 dilution, and Goat anti-Mouse IgG2a secondary antibody conjugated to Alexa Fluor 546 (ThermoFisher, catalog number A-21133) was used at a 1:500 84336) was used at a 1:100 dilution, and Goat anti-Rabbit IgG secondary antibody conjugated to Cy3 (Jackson Immunoresearch, catalog number 111-165-003) at a 1:450 dilution.

Platelet Releasates. Platelet releasates were generated from expired human platelet units provided by the University of Michigan Health Pathology Blood Bank. Platelet units were washed twice in freshly made calcium free Tyrode’s buffer with centrifugations at 1000 x g for 10 minutes at 25°C. Washed platelets were diluted to 1 x 10⁹ cells / ml in Tyrode’s buffer warmed to 37°C containing 5 µM PAR-1 selective activating peptide (Sigma Aldrich, catalog number S1820) to activate platelets. The suspension was agitated at 75 rpm for 20 minutes in a 37°C shaker. Platelets were removed by centrifugation at 3000 x g for 10 minutes and resulting supernatant of activated platelet releasates were diluted to 300 µg/ml of total protein content. NIH-3T3 cells were stimulated with 0.05% platelet releasates in DMEM containing 1.5% serum for 48 hours.

Production of anti-polyphosphate hybridomas. Anti-polyphosphate hybridomas were produced over individual fusions utilizing the autoimmune mouse strains NZBWF1/J and MRL/MPJ-Fas<lpr>/J both from Jackson Labs. A mix of males and females of both strains were either non-immunized or immunized with 250 µg DNA (Sigma) plus l00 µg histones (Roche) both from calf thymus with Freund’s Complete Adjuvant and then boosted with the same in phosphate buffered saline. The most productive fusion was from a non-immunized NZBWF1/J female aged 9 months at the time of splenocyte harvest and fusion with the mouse myeloma P3X63AG8-653. This myeloma cell line was used for all of the fusions. Numerous anti-polyphosphate monoclonal antibody producing hybridomas were derived from this fusion. Hybridomas were produced from standard culture and testing methodologies and expanded in serum free media for monoclonal antibody production and purification.

For the rat renal ischemia-reperfusion data, magnetic resonance imaging (MRI) and spectroscopy (MRS) were used to assess renal morphology, measured by changes in MRI signal intensities (SI), vascular alterations, measured by obtaining tissue vascular perfusion rates which were calculated as relative tissue blood flow (rTBF), and metabolite changes, by measuring levels of trimethylamines (TMA) and lipids. It was found that the ischemia- reperfusion injury (IRI) had significantly decreased MRI SI, significantly decreased rTBF, and significantly increased TMA levels, compared to the control (normal) kidney, whereas polyp antibody therapy had restored morphology, rTBF and TMA levels, all back to normal conditions. In particular, the morphological assessments (both MRI and histology), as well as seemed to circumvent. A decrease in MRI SI following IRI indicates that the tissue has had significant damage, which includes necrosis, as well as early stages of fibrosis. A decrease in vascular perfusion either indicates damage to the tissue vascularity and/or tissue damage (e.g., necrosis and early fibrosis) that restricts vascular blood flow.

FIGS. 3A-C show that platelet-sized polyphosphate induces myofibroblast differentiation. FIGS. 4A-B show that platelet-sized polyphosphate increases collagen production. FIGS. 5A-B show that platelet releasates induce myofibroblast differentiation. FIGS. 6A-B show that Poly-P antibody restores kidney morphology in rat renal ischemia- reperfusion model. FIGS.7A-D show that Poly-P antibody restores kidney morphology in rat renal ischemia-reperfusion model. FIG. 8 shown that Poly-P antibody restores kidney morphology in rat renal ischemia-reperfusion model. FIGS.9A-D show that Poly-P antibody restores kidney metabolites in rat renal ischemia-reperfusion model. FIG.10 shows that Poly- P antibody restores kidney metabolites. Kidney metabolites TMA (trimethylamines) are significantly elevated in ischemia reperfusion injury (Serkova, 2005). FIGS.11A-B show that Poly-P antibody restores kidney vascular perfusion rates to normal in rat renal ischemia- reperfusion model. FIG.12 shows that Poly-P antibody restores kidney vascular perfusion to normal. Kidney perfusion rates are significantly reduced in ischemia reperfusion injury, both in the cortex and medulla regions. FIG. 13 shows that Polyphosphate enhances fibroblast migration. * * * * * * * * * * * * * * * *

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps or in the sequence of steps of the method described herein, without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference: U.S. Pat. No.3,817,837

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Claims

1. A method of inhibiting a fibrosis or a fibrotic condition involving extracellular polyphosphate toxicity in a subject comprising administering to said subject a polyphosphate-degrading enzyme.

2. The method of claim 1, wherein said fibrotic condition is fibrosis of lung, liver, heart, brain, artery, knee, shoulder, intestine, hands, fingers, kidney, uterus, vagina, gall bladder, bile duct, skin, mediastinum, bone marrow, penis, or soft tissue.

3. The method of claim 1, wherein said fibrotic condition comprises pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced injury, such as radiation-induced lung injury, chemotherapy-induced fibrosis, cirrhosis of the liver, atrial fibrosis, endomyocardial fibrosis, old myocardial infarction, glial scar, arterial stiffness, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, adhesive capsulitis or trauma.

4. The method of claims 1-3, wherein said subject is selected from a group consisting of human, dog, cat, horse, monkey, mouse, rat, rabbit, sheep, goat, cow, and pig.

5. The method of claims 1-4, wherein the polyphosphate-degrading enzyme is bacterial exopolyphosphatase, such as PPX, a yeast exopolyphosphatase, such as ScPPX1, or an endopolyphosphatase.

6. The method of claims 1-5, further comprising administering to said subject a second anti-fibrosis therapy.

7. The method of claim 6, wherein the second anti-fibrosis therapy is an anti-histone antibody.

8. The method of claims 1-7, further comprising assessing polyphosphate content in a serum or plasma sample from said subject before or after administering said antibody. antibody, and said method further comprises adjusting a dosage of said administered anti-polyphosphate antibody responsive to said extracellular polyphosphate content. 10. The method of claim 8, wherein assessing is performed before administering said polyphosphate-degrading enzyme. 11. The method of claim 8, wherein the assessing comprises ELISA or Western blotting using anti-polyphosphate antibodies. 12. A method of inhibiting a fibrosis or a fibrotic condition involving extracellular polyphosphate toxicity in a subject comprising administering to said subject an anti- polyphosphate antibody or antigen-binding fragment thereof. 13. The method of claim 12, wherein said fibrotic condition is fibrosis of lung, liver, heart, brain, artery, knee, shoulder, intestine, hands, fingers, kidney, uterus, vagina, gall bladder, bile duct, skin, mediastinum, bone marrow, penis, or soft tissue. 14. The method of claim 12, wherein said fibrotic condition comprises pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced injury, such as radiation-induced lung injury, chemotherapy-induced fibrosis, cirrhosis of the liver, atrial fibrosis, endomyocardial fibrosis, old myocardial infarction, glial scar, arterial stiffness, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, adhesive capsulitis or trauma. 15. The method of claims 12-14, wherein said subject is selected from a group consisting of human, dog, cat, horse, monkey, mouse, rat, rabbit, sheep, goat, cow, and pig. 16. The method of claims 12-15, wherein said anti-polyphosphate antibody is a monoclonal antibody. 17. The method of claims 12-15, wherein said anti-polyphosphate antibody is comprised in a polyclonal antisera, such as purified polyclonal antisera. recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab)₂ fragment, or Fv fragment. 19. The method of claims 12-15, wherein said anti-polyphosphate antibody is a chimeric antibody or is bispecific antibody. 20. The method of claims 12-19, wherein said anti-polyphosphate antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. 21. The method of claims 12-20, further comprising administering to said subject a second anti-fibrosis therapy. 22. The method of claim 21, wherein the second anti-fibrosis therapy is an anti-histone antibody or a polyphosphate-degrading enzyme. 23. The method of claims 12-22, further comprising assessing polyphosphate content in a serum or plasma sample from said subject before or after administering said antibody. 24. The method of claim 23, wherein assessing is performed after administering said antibody, and said method further comprises adjusting a dosage of said administered anti-polyphosphate antibody responsive to said extracellular polyphosphate content. 25. The method of claim 23, wherein assessing is performed before administering said antibody. 26. The method of claim 23, wherein the assessing comprises ELISA or Western blotting using anti-polyphosphate antibodies.