AU2017200039A1 - Arthritis treatment - Google Patents

Abstract

Administration of a monoclonal Ab (mAb) that specifically targets IL-la is useful to treating articular and extra-articular symptoms of arthritis.

Description

ARTHRITIS TREATMENT 2017200039 04 Jan 2017

CROSS-REFERENCE TO RELATED APPLICATIONS

[0000] The present application is a divisional application of Australian Application No. 2015203524, which is incorporated in its entirety herein by reference.

[0001] The present application claims the priority of U.S. provisional patent application serial number 61/356,176 filed on June 18, 2010, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates generally to the fields of immunology, inflammation, arthritis, and medicine. More particularly, the invention relates to the use of antibodies (Abs) which specifically bind interleukin-la (IL-la) to treat one or more symptoms of arthritis.

BACKGROUND

[0003] Arthritis, the most common cause of disability in the United States, is a collection of different conditions such as osteoarthritis, rheumatoid arthritis, gout, psoriatic arthritis, septic arthritis, and reactive arthritis. All types of arthritis are characterized by joint inflammation which causes pain, swelling, redness, stiffness, and warmth at that affected site. Because afflicted subjects are less mobile due to pain and stiffness, arthritis can indirectly lead to obesity, high cholesterol, and/or heart disease. Arthritis can also cause extra-articular disease such as iritis, uveitis, oral ulcers, inflammation of the gastrointestinal tract, inflammation of the genitourinary tract, and skin lesions.

[0004] For most types of arthritis, no cure exists and treatment is largely symptomatic, e.g., administration of analgesics and anti-inflammatory drugs. Non-steroidal anti-inflammatory drugs (NSAIDs) can be used to reduce inflammation and pain. While generally effective, NSAIDs may cause side effects such as abdominal pain, bleeding, ulcers, and liver and kidney damage. Corticosteroids are effective at reducing inflammation and joint damage, but can cause a number of side effect are also associated including bruising, weight gain, cataracts, bone thinning, diabetes, and hypertension. Other drugs commonly used to treat arthritis are methotrexate, cyclosporine, cyclophosphamide, leflunomide, hydroxychloroquine, sulfasalazine, and minocycline. These too can cause side effects such as liver damage and - 1 - 2017200039 04 Jan 2017 immunosuppression. Tumor necrosis factor (TNF) inhibitors like etanercept (Enbrel), infliximab (Remicade), and adalimumab (Humira) are also useful for treating arthritis. - la -

Side effects of TNF inhibitors include injection site reactions, heart failure, lymphoma, and increased risk of infection. 2017200039 04 Jan 2017

SUMMARY 100651 The invention is based on the discovery that administration of an antibody (Ab) that specifically targets IL-la in a human subject suffering from arthritis reduces the number of CD14-HL-la+ peripheral blood monocytes in the subject and markedly ameliorates inflammation in both articular and extraarticular sites ~ all without any observed side effects other than pain at the administration site.

[0000) Accordingly, the invention features a method of treating an inflammatory pathology associated with arthritis in a human subject by administering to the subject a pharmaceutical composition including a pharmaceutieaily acceptable carrier and an. amount of an anti-lL-la antibody effective to reduce at least one symptom of the inflammatory pathology in the subject. The symptom can be joint inflammation such as of the wrist or shoulder, or inflammation of the eye such as uveitis. The anti-IL-Icx antibody can be a monoclonal antibody such as an IgGl. The aati-IL-ia antibody can be the monoclonal antibody designated as MABpl or a monoclonal antibody that includes one or more complementarity determining regions (CDRs) of MABpl.

[00071 The pharmaceutical composition can be administered to the subject by injection, subcutaneously, intravenously, intramuscularly, intraoeuiariy, or directly into an inflamed joint. The antibody might also be administered to the eye topically. In the method, the amount of the anti-lL-ία antibody effective to reduce at least one symptom of the inflammatory pathology in the subject can be sufficient to raise the subject’s peripheral blood concentration of anti-IL-Ia antibody to at least 4 ug/ml; and/or sufficient to decrease the number of the subject’s CD14+IL-I.a + peripheral blood monocytes by at least 5%.

[00081 The method might also include a step of measuring the number of CDi4+IL-lo.? monocytes in the subject’s peripheral blood after administration of the pharmaceutical composition, e.g., wherein the step of measuring the number of C014i-lL-la+ monocytes in the subject’s peripheral blood is performed at least two different time points after administration of the pharmaceutical composition.

[00691 In another aspect, the invention features a method inducing monocyte vacuolization in a subject by administering to the subject a pharmaceutical 2 composition including a pharmaceutically acceptable earner and an amount of an anti-IL-la antibody effective to induce vacuole formation in monocytes. 2017200039 04 Jan 2017 10018| Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill In the art to which this invention belongs. Commonly understood definitions of biological terms can be found in Rieger et al„ Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994, Commonly understood definitions of medical terms can be found in Stedman’s Medical Dictionary1, 27* Edition, Lippincott, Williams & Wilkins, 2000, [0011] As used herein, an “antibody” or “Ab” is an immunoglobulin (Ig), a solution of identical or heterogeneous Igs, or a mixture of Igs. An “antibody·5 can also refer to fragments and engineered versions of igs such as Fab, Fab5, and F(ab5)> fragments; and scFv’s, heteroconjugate Abs, and similar artificial molecules that employ Ig-derived CDRs to impart antigen specificity. A “monoclonal antibody” or “mAh” is an Ab expressed by one clonal B cell line or a population of Ab molecules that contains only one species of an antigen binding site capable of immunoreacting with a particular epitope of a particular antigen. A “polyclonal antibody” or “polyclonal Ab55 Is a mixture of heterogeneous Abs, Typically, a polyclonal Ab will include myriad different Ab molecules which bind a particular antigen with at least some of the different Abs immunoreacting with a different epitope of the antigen. As used herein, a polyclonal Ab can be a mixture of two or more mAbs, 100121 An “antigen-binding portion” of an Ab is contained within the variable region of the Fab portion of an Ab and is the portion of the Ah that confers antigen specificity to the Ab (I*?., typically the three-dimensional pocket formed by the CDRs of the heavy and light chains of the Ab), A "Fab portion" or "Fab region" is the proteolytic fragment of a papain-digested Ig that contains the antigen-binding portion of that Ig, A "non-Fab portion" is that portion of an Ab not within the Fab portion, e,g„ an “Fc portion*5 or “Fc region,55 A “constant region'5 of an Ab is that portion of the Ab outside of the variable region. Generally encompassed within the constant region is the “effector portion5' of an Ab, which is the portion of an Ab that is responsible for binding other immune system components that facilitate the immune response. Thus, for example, the site on an Ab that binds complement components or Fc receptors (not via its antigen-binding portion) is an effec tor portion of that Ab. 3 [0013j When referring to a protein molecule such as an Ab, “purified” means separated from components that naturally accompany such molecules. Typically, an Ab or protein is purified when it is at. least about 10% (e.g., 9%, 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, and 100%), by weight, tree from the non-Ab proteins or other naturally-occurring organic molecules with which it is naturally associated. Purity can be measured by any appropriate method, e.g., eolonrn chromatography, polyacrylamide gel electrophoresis, or HPLC analysts. A chemically-synthesized protein or other recombinant protein produced in a cell type other than the cell type in which it naturally occurs is “purified.” 2017200039 04 Jan 2017 (00141 By “bind”, “binds” or “reacts with” is meant that one molecule recognizes and adheres to a particular second molecule in a sample, but does not substantially recognize or adhere to other molecules in the sample. Generally, an Ab that "specifically binds” another molecule has a greater than about 10s, 106, 10', 10s, lOy ΙΟ10, 10u, or 10° liters/mole for that other molecule. (00151 A "therapeutically effective amount" is an amount which is capable of producing a medically desirable effect in a treated animal or human (e.g., amelioration or prevention, of a disease or symptom of a disease), [00161 Although methods and materials similar or equivalent to those described herein can be used in the practice or tearing of the present invention, suitable methods and materials are described below. In addition, the particular embodiments discttsscd below are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS {00171 Figure 1 is a graph and table showing the pharmacokinetics of MABpl after administration to a human subject with reactive arthritis. (001.81 Figure 2 is a series of graphs and histograms showing flow cytometric blood analyses after administration of MABpl to a human subject with reactive arthritis. [0019| Figure 3 is a series of graphs showing flow cytometric blood analyses after administration of MABpl to a human subject with reactive arthritis.

DETAILED DESCRIPTION (0020) The invention encompasses compositions and methods for treating a symptom or pathologic process associated with arthritis in a subject The below described prefened embodiments illustrate adaptation of these compositions and methods. 4

Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below. 2017200039 04 Jan 2017

General Methodology [00211 Methods involving conventional immunological and molecular biological techniques are described herein. Immunological methods (for example, assays for detection and localization of antigen-Ab complexes, immunoprecipitation, immunoblottmg, and the like) are generally known in the art and described in methodology treatises such as Current Protocols in .Immunology, Coligan et al, ed., Mm Wiley & Sons, New York, Techniques of molecular biology are described in detail in treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Sambroak. et al, ed,, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, K.Y., 2001; and Current Protocols in Molecular Biology, Ausubel et al, ed., Greene Publishing and Wiley-Interscience, New York, Ab methods are described in Handbook of Therapeutic Abs, Dubcl, S., ed., Wiley-VCli 2007. General methods of medical treatment am described in McPhee and Papadakis, Current Medical Diagnosis and Treatment 2010, 49l)* Edition, McGraw-Hill Medical, 2010; and Fauci et al, Harrison’s Principles of internal Medicine, 17th Edition, McGraw-Hill Professional, 2008

Treatment of Arthritis Symptoms {0022) The compositions and methods described herein are useful for treating ait inflammatory pathology associated with arthritis in a mammalian subject by administering to the subject a pharmaceutical composition including an amount of an anti-IL-Ια antibody effective to reduce at least one symptom of the inflammatory pathology in the subject. The mammalian subject might be- any that suffers from arthritis including, human beings, dogs, cats, horses, cattle, sheep, goats, and pigs. Human subjects might he male, female, adults, children, seniors (65 and older), and those with other diseases. The particular symptom or pathologic process associated with arthritis can be inflammation, pain, stiffness, or degeneration of a joint (e.g,, in the wrist, digits [metacarpal or metatarsal joints], elbows, shoulders, hips, knees, ankles, foot, neck, or back) or extraartieular tissue (e.g., iritis, uveitis, oral ulcers, inflammation of the gastrointestinal tract, inflammation of the genitourinary tract, or skin lesions).

Antibodies and other Agents that Target IL-l a 5 {0023] Any suitable type of Ab that specifically binds XL-la and reduces a symptom or pathologic process caused by arthritis in a subject might be used in the invention. For example, the anti-IL-la Ab used might be mAb, a polyclonal Ab, a mixture of mAbs, or an Ab fragment or engineered Ab-1 ike molecule such as an scFv. The Ka of the Ab is preferably at least 1 xlO* M~l or greater (e.g., greater than 9 xl0!fe M"1, 8 x!0w M4, 7 xlO10 M4 6 χ10ί0 M4, 5 xlO10 M4,4 xlO10 M4, 3 xlOi0 M'\ 2 xlOf,> M' *, or 1 x.1010 M4). In a preferred embodiment, the invention utilizes a folly human mAb that includes (i) an antigen-binding variable region that exhibits very high binding affinity for human IL-laand (ii) a constant region that is effective at both activating the complement system though Clq binding and binding to several different Fc receptors. The human Ab is preferably an IgG i. although it might be of a different isotvpe such as IgM, IgA, or IgE, or subclass such as IgG2, IgG3, or IgG4, One example of a particularly useful mAb is MABpi, an IL-la-specific IgG! monoclonal antibody described in U.S. patent application serial number 12/455,458 filed on June 1, 2009. Other useful mAbs are those than include at least one but preferably all the CDRs ofMABpl. 2017200039 04 Jan 2017 {0024] Because B lymphocytes which express Ig specific for human IL-Ια occur naturally in human beings, a presently preferred method for raising mAbs is to first isolate such a B lymphocyte from a subject and then immortalize it so that it can be continuously replicated in culture. Subjects lacking large numbers of naturally occurring B lymphocytes which express Ig specific for human IX-la may be immunized with one or more human 11,- l a antigens to increase the number of such B lymphocytes. Human mAbs are prepared by immortalizing a human Ab secreting cell (e.g., a human plasma cell). See, e.g., U.S. patent no. 4,634,664. jO02S| In an exemplary method, one or more (e.g., 5, 10, 25, 50, 100, 1000, or more) human subjects are screened for the presence of such human 'LL-l a-specific Ab in their blood. Those subjects that express the desired Ab can then be used as B lymphocyte donors, in one possible method, peripheral blood is obtained from a human donor that possesses B lymphocytes that express human IL-la-specific Ab, Such B lymphocytes are then isolated from the blood sample, e.g., by cells sorting (e.g,, fluorescence activated cell sorting, "FACS”; or magnetic bead cell sorting) to select B lymphocytes expressing human IL-la-specific ig. These cells can then be immortalized by viral transformation (e.g,, using ESV) or by fusion to another 6 immortalized cell such as a hitman myeloma according to known techniques. The B lymphocytes within this population that express Tg specific for human IL-lacan then be isolated by limiting dilution methods (e.g., cells in wells of a microtiter plate that are positive for Ig specific tor human IL-ία are selected and subcultured, and the process repeated until a desired clonal line can be isolated). See, e,g.. Coding, Monoclonal Abs: Principles and Practice, pp. 59-103, Academic Press, 1986. Those clonal cell lines that express Ig having at least nanomolar or pieomolar binding affinities for human IL-ί α are preferred. MAbs secreted by these clonal cell lines can be purified from the culture medium or a bodily fluid (e.g., ascites) by conventional Ig purification procedures such as salt cuts, size exclusion, ion exchange separation, and affinity chromatography, 2017200039 04 Jan 2017 10026| Although immortalized B lymphocytes might be used in m vhm cultures to directly produce mAbs., in certain cases it might be desirable to use heterologons expression systems to produce mAbs, See, e,g„ the methods described in U.S. patent application number 11/754,899. For example, the genes encoding an mAb specific for human IL-la might be cloned and introduced into an expression vector (e.g., a plasmid-based expression vector) for expression in a heterologous host ceil (e.g„ CEO cells, COS cells, myeloma cells, and E. cola cells). Because Igs include heavy (H) and light (L) chains in an 1-1¾ configuration, tire genes encoding each may be separately isolated and expressed in different vector's. |0027] Although generally less preferred due to the pester likelihood that a subject will develop an anti-Ab response, chimeric mAbs (e.g., “humanized” mAbs), which are antigen-binding molecules having different portions derived from different animal species (e.g., variable region of a mouse Ig fused to the constant region of a human Ig), might be used in the invention. Such chimeric Abs can be prepared by methods known in the art. See, e.g., Morrison et aL Free. Natl. Acad. Set. USA, 81:6851, 1984; Neuberger et al, Nature, 312:604, 1984; Takeda et al, Nature, 314:452, 1984. Similarly, Abs can be humanized by methods known in the art For example, monoclonal Abs with a desired binding specificity can be humanized by various vendors or as described, in U.S. Pat. Nos. 5,693,762; 5,530,101; or 5,585,089. {002$! The mAbs described herein might be affinity matured to enhance or otherwise alter their binding specificity by known methods such as VH and VL domain shuffling (Marks et al. Bio/Technology 10:779-783, 1992), random mutagenesis of 7 the hypervariable regions (HVRs) and/or framework residues (Barbas et al. Proc Nat. Acad. Set USA 91:3809-3813,1994; SeMer et al Gene 169:147-155,1995; Yelton et al, I. Immunol. 155:1994-2004, 1.995; Jackson et al, J, Immunol. 154(7):3310-9, 1995; and Hawkins et al, j. Mol Biol. 226:889-896. 1992. Amino acid sequence variants of an Ab may be prepared by introducing appropriate changes into the nucleotide sequence encoding the Ab, In addition, modifications to nucleic acid sequences encoding mAbs might be altered (e.g., without changing the amino acid sequence of the mAh) for enhancing production of the mAh in certain expression systems (e.g,, intron elimination and/or codon optimization for a given expression system). The mAbs described herein can also be modified by conjugation to another protein (e.g,, another mAh) or non-protein molecule. For example, a mAb might be conjugated to a water soluble polymer such as polyethylene glycol or a carbon nanotube (See, e.g,, Kam et al., Proc. Natl. Acad, Set, USA 102: 11600-11605, 2005). See, U.S. patent application number 11/754,899. 2017200039 04 Jan 2017 {99291 Preferably, to ensure that high titers of human IL-Ια "Specific mAb can be administered to a subject wi th minimal adverse effects, the mAb compositions of the invention are at least 0.5, 1, 2, 3,4,5,6,7, 8, 9, 10., II, 12, 13, 14,15,20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 99.9 or more percent by weight pure (excluding any excipients). The mAb compositions of the invention might include only a single type of mAh (i.e., one produced from a single clonal .B lymphocyte line) or might include a mixture of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different types of mAbs, 19930] To modify or enhance their function, the human IL-Ια mAbs might be conjugated another molecule such as a cytotoxin. A human XL-ία. specific mAb might be conjugated with one or more cytotoxins to more effectively kill cells expressing IL-Ια. Cytotoxins for use in the invention can be any cytotoxic agent (e.g., molecule that can kill a cell after contacting the ceil) that can be conjugated to a human IL-la specific mAb, Examples of cytotoxins include, without limitation, radionuclides (e.g.. >S, ,4C, mY, *»Zr, ®Ti( ^Re, mRe, 57Cu, mBi, and2»At), conjugated radionuclides, and chemotherapeutic agents, further examples of cytotoxins include, but are not limited to, antimetabolites (e.g., 5-fluorouriciI (5-Fll), methotrexate (MTX), fludarabine, etc.), anti-microtubule agents (e.g., vincristine, vinblastine, colchicine, taxanes (such as paclitaxel and docetaxel), etc.), alkylating agents (e.g., 8 cyclophosphamide, meiphalan, bischloroethylnitrosurea (BCNU), etc.}, platinum agents (e.g., cisplatin (also termed cDDP), carboplatin, oxaliplatin, JM-216, 0-973, etc.}, anthraeyclines (e.g., doxorubicin, daunorubicin, etc.), antibiotic agents (e.g., mitomvcin-C), topoisomerase inhibitors (e.g., etoposide, tenopostde, and camptothecins), or other cytotoxic agents such as rictn, diptheria toxin (DT), Pseudomonas exotoxin (PE) A, PE40, abrin, saporin, pokeweed viral protein, ethidium bromide, glucocorticoid, anthrax toxin and others. See, e.g., U.S. Pat. No. 5,932,188. 2017200039 04 Jan 2017 (0031} While the IL-ία specific Abs described above are preferred for use the invention, in some cases, other agents that specifically target IL-ία might be used so long as their administration leads to improvement of one or more symptoms of arthritis. These other agents might include small, organic molecules, aptamers, peptides, and proteins that specifically bind IL-l a.

Pharmaceutical Compositions and Methods (0032} The anti-IL-Ια Ab compositions may be administered to animals or humans in pharmaceutically acceptable carriers (e.g., sterile saline), that are selected on the basis of mode and route of administration and standard pharmaceutical practice. A list of pharmaceutical ly acceptable carriers, as well as pharmaceutical formulations, can be found in Remington’s Pharmaceutical Sciences, a standard text in this field, and in USP/NF. Other substances may be added to tire compositions and other steps taken to stabilize and/or preserve the compositions, and/or to facilitate their administration to a subject.

[0(133} For example, the Ab compositions might be fyophilized (see Draher et at, J, Immunol. Methods. 181:37, 1995; and PCT/US90/01383); dissolved in a solution including sodium and chloride ions; dissolved in a solution including one or more stabilizing agents such as albumin, glucose, maltose, sucrose, sorbitol, polyethylene glycol, and glycine; filtered (e.g., using a 0.45 and/or 0.2 micron filter); contacted with beta-propiolactone; and/or dissolved in a solution including a microhicide (e.g., a detergent, an organic solvent, and a mixture of a detergent and organic solvent.

[0034} The Ab compositions may be administered to animals or humans by any suitable technique. Typically, such administration will be parenteral (e.g., intravenous, subcutaneous, intramuscular, or intraperitoneal introduction). Tbe compositions may also be administered directly to the target site (e.g., an inflamed 9 joint, or the uvea or eonjuctiva) by, for example, injection or topical application. Other methods of delivery, e.g., liposomal delivery or diffusion from a device impregnated with the composition, are known in the art. The composition may be 2017200039 04 Jan 2017 administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously or by peritoneal dialysis), 1D035| A therapeutically effective amount is an amount which is capable of producing a medically desirable result in a treated animal or human. An effective amount of anti-IL-1 a Ab compositions is an amount which shows clinical efficacy in arthritis patients as measured by the improvement in pain and function as well as the prevention of structural damage. As is well known in the medical arts, dosage for anyone animal or human depends on many factors, including the subject’s size, body surface area, age, the particular composition to be administered, sex, time and route of administration, general health, and other drags being administered concurrently. A preferred dose is one that is sufficient to raise· the subject’s peripheral blood concentration of anti-IL-Ια Ab to at least 4 (e.g., at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,400, 500, 1000, 2500, or 5000) micrograms.mil. It is expected that an appropriate dosage of Abs would be in the range of about 0.2 to 20 (e.g., 0.5, 1,2, 3,4, 5, 6, 7, 8, 9, .10, .15, 20, 30,50, or 100) mg/fcg body weight for subcutaneous administration and about 0,001 to 50 (e.g., 0,001,0.01,1, 5,10,15, 25, or 50) mg per eye for topical administration to the eve. The dose may be given repeatedly, e.g., hourly, daily, weekly, or monthly.

EXAMPLES

Example 1 - Xilonsx™ 10036( Xilonix ™ is a sterile injectable liquid formulation of 15 mg/mL MABpi in a stabilizing isotonic buffer (pH 6.4). Each 10-mL Type 1 borosilicate glass serum vial contains 5 mL of the formulation, and is sealed with a 20-mm Daikyo Flurotec butyl rubber stopper and flip-off aluminum seal. The product is stored at 5±3°C, with excursions to room temperature permitted. The exact composition of the drag product is shown below:

(mm posit inn of the Drug Product (Xilonix™) ingredient Grade Manufacturer Concentration) MABpi antibody (AH' XBiotech 15 mg/mL 10 sodium phosphate dibasic compendial IT Baker 12 mg/mL citric acid monohydrafe compendial JT Baker 2 mg/mL Trehalose,2H20 (high-purity low endotoxin) compendial Perro- Pfanstiehl 60 mg/mL poiysorbate 80 compendial JT Baker 0.2 mg/mL Phosphoric acid, to adjust pH compendial JT Baker 0.04 mg/mL water for injection compendial Microbix q,s.

Example 2 - Treatment of Reactive Arthritis with an IL- kx-specific Monoclonal Antibody. 2017200039 04 Jan 2017 [0037] A 48 year-old male patient with reactive arthritis was administered a total 220 milligrams of MABpl, an IL~ la-specific monoclonal antibody described in li.S. patent application serial number 12/455,458 tiled on lane l, 2()09. The patient had a long history of reactive arthritis, starting at age 16, when tie was diagnosed with Reiter’s syndrome during hospitalization for severe inflammation in his left knee. This inflammation resolved, yet the patient experienced, periodic relapses in several joints until his mid-twenties. No further episodes occurred until, at age 35, the patient bad a severe unilateral episode of uveitis that lasted for 8 weeks. The uveitis was poorly managed with ophthalmic corticosteroids and oral MSAIDS, resulting in some scaring. The patient subsequently experienced at least three additional episodes of uveitis of varying intensities, one episode requiring subcorneal injection of corticosteroids.

[0038] Just prior to his 48th birthday, the patient developed severe pain in his left shoulder and wrist Evident swelling and redness with almost complete loss of mobility affected the wrist. The patient was unable to abduct Ms left arm greater than about 20° due to intense shoulder pain. On that day, the patient was given a subacromial injection of corticosteroids into die left shoulder. The patient reported that the condition continued to worsen with pain from shoulder and wrist reportedly becoming continuous, interrupting work and preventing sleep, In addition, pain and irritation in the left eye ensued, indicating onset of an episode of uveitis. This was reportedly the first time joint inflammation and uveitis occurred together. The patient 11 was taking ophthalmic corticosteroids, oral and topical ophthalmic 'NSAIDS with little apparent benefit. 2017200039 04 Jan 2017 jf)039j On day 0 (forty-two days after the subacromial injection of corticosteroids), the patient was administered four subcutaneous injections of MABpl, delivering a total of 110 mg of MABpl (in equal doses). No side effects other than pain during injection was reported. Blood was drawn by venous puncture immediately prior to injection into two $ ml sodium heparin tubes. Plasma analysis using an enzyme-linked immunadsorb&nt assay (ELISA) for the detection of existing endogenous anti-IL-la antibodies revealed no pre-existing antibodies. 10040] On day 1, the patient repotted that he woke up that morning without the throbbing pain that had become the “first sensation upon waking.” Over the next several days there was an evident improvement in mobility. There was no induration or redness at the injection sites. A blood draw was taken and .flow cytometric analysis (FAC'S) was performed to evaluate leuckocyte subsets and IL-Ια expression on monocytes. Analysis was also performed on plasma to determine levels of MABpl and to begin collection of pharmacokinetic (pK) data for MABpl, FACS analysis of PBMC revealed that most CD 14+ monocytes (72.6%) expressed IL-Ια. A MABpl plasma concentration of 3.2 pg/ml was observed. {0041] On day 6, another blood sample was taken and analyzed using FACS and for MABpl. The frequency of CD14+ monocytes stained by MABpl had declined to 47.3%. Plasma levels of MABpl had increased to 7 pg/ral. Although not confirmed, the increase in MABpl concentration was considered to reflect a depot effect of the subcutaneous administration of MABpl. Although there had been improvement, the patient still exhibited considerable tenderness and pain with movement and the uveitis had flared since the previous weekend, where the patient had attended a party and consumed alcohol. The patient was administered another ttOmg of MABpl subcutaneously. {0042] On day 14, a blood sample was taken and analyzed using FACS and pK analysts was performed on plasma. CD 14+ monocyte frequency stained by MABpl further declined to 21,7%. However, plasma levels of MABpl had also declined to S.SpgAul. This was unanticipated, since plasma levels of MABpl had increased over the week after the first injection. 12 i0O43j Approximately one month after the first injection of MABpl the patient was reevaluated. Marked improvement was noted in mobility and there was no pain in the wrist. Pain in shoulder was present only upon abduction to 90°. FACS analysis revealed no detectable CD 14+ monocytes stained by MABpl. Plasma levels of MABpl had declined to 1.6 ugrinl, suggesting a half-life for MABpl of about two weeks. 2017200039 04 Jan 2017 [00441 Over the course of the next several weeks the patient showed gradual but continuous improvement in mobility. There was complete resolution of the uveitis. The improvement was noted even though the patient discontinued use of all medications after the first injection of MABpl, Approximately three months after the first injection of MABpl. the frequency of CD14+ monocytes stained by MABpl had returned to pre-treatment levels. MABpl levels in plasma declined to 0.0? pg/ntl. However, the patient continued to do well with continuing improvement in mobility of the shoulder.

Example 3 - Screening of plasma samples for endogenous autoantibody against hlL-1A and Pharmacokinetics of MABpl [00451 A method was developed for the screening of plasma samples for endogenous autoantibody against human IL-la (hIL-la) using a direct ELISA, This method was also used to determine pharmacokinetics (pK) of MABpl after administration, with the exception, that higher dilutions plasma samples were made.

[00461 The direct ELISA involves coating of recombinant human IL-ία on a polystyrene microplate. The bound human 1'L-IA captures endogenous anti-human IL-la antibody from test samples. An HRP-eonjugated-Fc specific, mouse-anti-hiimao IgG is then used to detect the captured endogenous anti-human 1L-1A antibody, followed by treatment with TMB substrate. On reacting with. HRP enzyme, the TMB substrate produces a deep blue-colored soluble product. The enzymatic reaction is stopped by the addition of a stop solution that turns the blue-colored product to yellow. The colorimetric measurements are carried out on a micropiate reader at 450 nm.

[0047J About 5 ml plasma sample per sample is provided. Plasma is kept at 2-8*C prior to aiiquoting and storage at -80¾. Plasma samples are diluted 1:500, 1:1.000 and 1:2000 -fold to use as samples. A positive control in buffer is used containing 2ft 13 ug/ml MABpl antibody stock as .1:5,000 and 1:10,000-fold dilutions on microplate. Buffer is used as a negative control as well as a pre-determined negative control plasma, which is diluted as 1:1.,000, 1:2,000 and 1:5,000, An. additional positive plasma control is used, which is plasma spiked with 20 pg/ml MABpl antibody and diluted as 1:5,000 and 1:10,000 lor samples on the microplate. j004fi{ If the positive control value fells within ± 2 standard deviation, the ELISA data is considered acceptable. Ho wever i f the QC positive control value fal ls beyond ± 2 standard deviation, the ELISA data is considered unacceptable and the experiment would be repeated. Using a Kalcidagraph, the logarithmic mean absorbance of standard solution is plotted as a function of logarithmic concentration along with absorbance error bars. The standard curve should exhibit a linear behavior. Results from a pharmacokinetics analysis of samples taken, from the patient as described in Example 2 am shown in Fig, 1. 2017200039 04 Jan 2017

Example 4 ~ Flow cytometric (FACS) examination, of blood lineage subsets i0049| FACS procedures are described for both whole blood staining, and staining of peripheral blood mononuclear cells (PBMC) enriched from whole blood. Both whole blood and PBMC staining was performed on all samples. This FACS analysis allows relative percentage determination of blood lineage subsets: B and T lymphocytes, NK cells, monocytes, neutrophils, and .TL-ia+ cells. Results from FACs analyses of samples taken from the patient as described in Example 2 are shown in Figs. 2 and 3. A photomicrograph of a blood smear showed that MABpl administration caused extensive vacuolization in peripheral blood monocytes when analysed 32 days post administration.

Example 5 Treatment of Uveitis with an 1L-Ιά-speeific Monoclonal Antibody. 100501 About two months following resolution of the uveitis described in Example 2, the patient experienced another episode of uveitis (predominantly iritis). The patient was started on coriicoseroid and non-steroidal anti-inflammatory drops (NSAIDS). Oral NSA1DS were also used. The uveitis was unresponsive to treatment and progressed. However, there was no evidence of any joint involvement, with shoulder continuing to show improvement in mobility. The patient was administered MABpl topically to the affected eye. MABpl (I5mg/ml solution) was administered 14 at a rate of one drop per minute, for ten mirrutes, for a total of ten drops to the affected eye (approximately 3.75 mg in 0.25 mi). The patient did not complain of any pain during the administration. However, tor several hours after, die patient reported discomfort and burning. Oral NSAIDs were taken and the patient slept. The next morning, the patient reported considerable improvement, reduced pain and less inflammation than prior to administration. Twenty-four hours after the first administration of the MABPI drops, the patient administered 10 drops in the same fashion. Again, discomfort and burning was noted. Oral NSAIDs were taken, and again the patient took bed rest. The uveitis resolved itself completely. No further medications were taken. No recurrence of uveitis was observed over the next four months. 2017200039 04 Jan 2017

Other Embodiments [00511 It is to be undemood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the foll owing c laims.

What is claimed is; 15

Claims (10)

1. Claims

   1. Use of an anti-IL-Ια antibody to treat at least one symptom of an inflammatory pathology associated with arthritis in a human subject.
2. 2. The use according to claim 1, wherein the at least one symptom is joint inflammation.
3. 3. The use according to claim 1, wherein the at least one symptom is inflammation of the eye.
4. 4. The use according to claim 3, where in the inflammation is uveitis.
5. 5. The use according to claim 1, wherein the anti-IL-Ια antibody is a monoclonal antibody.
6. 6. The use according to claim 5, wherein the monoclonal antibody is an IgGl.
7. 7. The use according to claim 5, wherein the monoclonal antibody comprises a complementarity determining region of MABpl.
8. 8. The use according to claim 5, wherein the monoclonal antibody is MABpl.
9. 9. The use according to claim 1, wherein the anti-IL-Ια antibody is formulated in a pharmaceutical composition suitable for administration by injection.
10. 10. The use according to claim 1, wherein the anti-IL-Ια antibody is formulated in a pharmaceutical composition suitable for topical administration to the eye.