WO2014037785A2

WO2014037785A2 - Catalytic antibodies and uses thereof

Info

Publication number: WO2014037785A2
Application number: PCT/IB2013/001908
Authority: WO
Inventors: Rostyslav Bilyy; Eugenia BILA; Yuriy KIT
Original assignee: Institute Of Cell Biology, National Academy Of Sciences Of Ukraine
Priority date: 2012-09-06
Filing date: 2013-09-05
Publication date: 2014-03-13
Also published as: WO2014037785A3; UA105278C2

Abstract

The invention provides methods for making catalytically active antibodies (abzymes) having sialidase activity using neuraminidase inhibitors as antigens and methods for using the abzymes to treat autoimmune diseases and cancer.

Description

CATALYTIC ANTIBODIES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from Ukrainian Patent Application No. a201210528, filed on September 6, 2012, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

[0002] The invention provides novel abzymes that have sialidase activity. These abzymes may be used to treat disease-states resulting from decreased clearance of apoptotic cells and/or to treat cancer by targeting cancer cells so that the sialidase activity of the abzyme cleaves sialic acid on cancer cells, marking the cancer cells for clearance and/or cell death.

BACKGROUND OF THE INVENTION

[0003] All publications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Abzymes are antibodies that exhibit catalytic properties. These antibodies may be isolated from the blood serum of patients with various diseases (natural abzymes) [Paul, et ah, Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody. Science (1989), 244: 1 158-62], or they may be synthetic (induced) abzymes obtained by immunizing animals with conjugates of low-molecular-weight synthetic analogues (haptens) of compounds that imitate the transitional state of a certain chemical reaction with a high- molecular- weight polymeric carrier [Tramontano et al. Catalytic antibodies. Science, (1986), 234.1570-3].

[0005] Known catalytically-active antibodies with sialidase activity separated from the serum of patients with diseases are associated with the hyper-functioning of B-lymphocytes in some diseases, particularly multiple myeloma [Bilyy et al., Antibody-mediated sialidase activity in blood serum of patients with multiple myeloma. J Mol. Recognit, 2011. 24. 576-84] and systemic lupus erythematous [Bilyy et al., Cell surface glycans at SLE: changes during cell death, utilization for disease detection and molecular mechanism underlying their modification. Autoimmune Disorder - Pathogenic Aspects, Dr. Clio Mavragani (Ed.), 201 1 89-1 10 ISBN:978-953-307-643-0]. These abzymes are capable of desialating a number of biological targets, among which are gangliosides, the membrane glycoprotein(s) of erythrocytes, and the glycocalyx of eukaryotic cells. Desialation of the hydrocarbon determinants of apoptotic cells is capable of exacerbating their phagocytosis, which can serve as a protective mechanism for the occurrence of some autoimmune diseases in humans [Meesman, H. et al., Decrease of sialic acid residues as an eat-me signal on the surface of apoptotic lymphocytes. J. Cell Sci, 2010. 123:3347-3356], and it can become a potential approach to the therapy of autoimmune diseases [Bilyy et al., Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles. J Biol. Chem, 2012 287:496- 503; Munoz et al., The role of defective clearance of apoptotic cells in systemic autoimmunity. Nat. Rev. Rheumatol, 2010 6:280-289]. Since abzymes with sialidase activity are present in the blood serum of persons with autoimmune and oncohematologic diseases, the therapeutic use of antibody preparations purified from the blood serum of patients is clinically risky and inadequate, with respect to bioethical considerations. The use of viral and bacterial neuraminidases is limited in connection with their immunogenicity and high pathogenicity [Kim et al., Features and applications of bacterial sialidases Applied Microbiology and Biotechnology 201 1 91 : 1-15; Shtyrya et al, Influenza virus neuraminidase: structure and function. Acta Naturae, 2009 1 :26-32], and the use of human recombinant sialidases is limited due to their instability during storage and their high production costs [Miyagi and Yamaguchi, Mammaliam sialidases: Physiological and pathological roles in cellular functions. Glycobiology 2012 22:880-896].

[0006] Herein, inventors provide abzymes having sialidase activity that may be used to treat disease-states resulting from decreased (reduced, in efficient) clearance of apoptotic cells and/or to treat cancers. SUMMARY OF THE INVENTION

[0007] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0008] Provided herein are methods for producing abzymes having sialidase activity by using an inhibitor of neuraminidase as an antigen and immunizing an animal with the antigen so as to produce the abzymes having sialidase activity. The inhibitor, which forms the antigen, may further comprise a bridging group and a carrier. The inhibitor may inhibit any one or more of Neul, Neu2, Neu3 and/or Neu4.

[0009] Further provided herein is an abzyme or a derivative or a fragment thereof having sialidase activity. In some embodiments, the abzyme is selected from the group consisting of monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, chimeric antibodies, humanized antibodies, human antibodies and a single chain antibody.

[0010] Also provided herein are methods for using the abzymes, for example, abzymes produced by the methods described herein, to treat, inhibit, reduce the severity of and/or promote prophylaxis of autoimmune diseases in a subject in need thereof. The methods include administering to the subject an effective amount of a pharmaceutical composition comprising the abzyme. In some embodiments, additional therapeutic agents may be used in conjunction with the composition described herein so as to treat, inhibit, reduce the severity of and/or promote prophylaxis of autoimmune diseases in the subject.

[0011] Further provided herein are methods for using the abzymes, for example, abzymes produced by the methods described herein, to treat, inhibit, reduce the severity of and/or promote prophylaxis of cancer in a subject in need thereof. The methods include administering to the subject an effective amount of a pharmaceutical composition comprising the abzyme. In some embodiments, additional therapeutic agents may be used in conjunction with the composition described herein so as to treat, inhibit, reduce the severity of and/or promote prophylaxis of cancer in the subject. [0012] Also provided herein are pharmaceutical compositions and kits comprising an abzyme having sialidase activity, for example the abzymes produced by the methods described herein. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF FIGURES

[0013] Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0014] Figure 1 depicts, in accordance with various embodiments of the present invention, a schematic representation of the conjugation of the sialidase inhibitor DANA with a carrier protein. DANA is 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, a neuraminidase inhibitor that contains a carboxyl group, EDC is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, and R is a high-molecular-weight carrier, for instance a protein molecule.

[0015] Figure 2 depicts, in accordance with various embodiments of the present invention, a schematic representation of purification of IgG and F(ab)2 antibodies produced by methods described herein.

[0016] Figure 3 depicts, in accordance with various embodiments of the present invention, the purity of the IgG antibodies produced by the methods described herein. Abzymes obtained after purification with ammonium sulphate precipitation, protein-G affinity chromatography and HPLC-SEC are essentially pure as can be seen for rabbit abzymes in (A) further fractionated using HPLC-SCX in the NaCl gradient and subsequent measurement of sialidase activity of each fraction; they also are not contaminated with immune complexes as demonstrated for SLE abzyme in (B) during HPLC-SEC at acidic conditions, pH 2.6.

[0017] Figure 4 depicts, in accordance with various embodiments of the present invention, the enzymatic activity of catalytically active antibodies (abzymes) with sialidase activity obtained by the methods described herein. (A) depicts the sialidase activity in (1) serum from control rabbits, (2) Ig fraction of immunized rabbits and (3) class-IgG fraction of immunized rabbits. (B) depicts the kinetic properties of the sialidase reaction catalyzed by purified class- IgG immunoglobulins obtained from immunized rabbits by the methods described herein, Michaelis-Menten graph. (C) depicts the kinetic properties of the sialidase reaction catalyzed by purified class-IgG immunoglobulins obtained from immunized rabbits by the methods described herein, Lineweaver-Burk plot.

[0018] Figure 5 depicts, in accordance with various embodiments of the present invention, analysis of the content of mixed sialyl glycoconjugates on the surface of leukemia T-cells of the human Jurkat line, intact and apoptotic (irradiated with ultraviolet UVB light, 180 M J/cm² for 14 hours before beginning the experiment): unprocessed, processed with catalytically active antibodies with sialidase activity and with neuraminidase. In all cases, the processing is accomplished with a sample with an activity of 30 mU (30 nmoles/min) over four hours.

[0019] Figure 6 depicts, in accordance with various embodiments of the present invention, the enzymatic activity of catalytically active antibodies (abzymes) and fragments thereof with sialidase activity obtained by the methods described herein. After precipitation with ammonium sulphate and affinity chromatography on protein-G sepharose, IgG preparation from patients with SLE (top row) or from immunized rabbits (bottom row) were further purified by HPLC SEC and the shaded fraction was collected (A) and used to prepare F(ab)2 fragments, subjected to PAGE in denaturing and non-denaturing conditions (B) as well as tested for the presence of sialidase activity and its inhibition with DANA (C).

[0020] Figure 7 depicts, in accordance with various embodiments of the present invention, that the sialidase abzymes produced by the methods described herein significantly facilitate clearance of viable and apoptotic prey (polymorphonuclear leukocytes (PMN) cells) by human monocyte-derived macrophages (MoMa). Prey cells were pre-treated with indicated desialylating agents and co-incubated with macrophages, afterward uneaten cells were counted. (A) human autologous model (PMN+MoMa), treatment with sialidase abzymes from patients with SLE blood. (B) human allogeneic model (PMN+MoMa), treatment with sialidase-abzymes from immunized rabbits.

DETAILED DESCRIPTION OF THE INVENTION

[0021] All references cited herein, including the references cited therein, are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are fully explained in the literature. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, NY 2001), Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken NJ, John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd Edition 2005), Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-3, 4-2), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, (2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3) provide one skilled in the art with a general guide to many of the terms used in the present application.

[0022] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0023] "Autoimmune diseases" as used herein include but are not limited to Acute disseminated encephalomyelitis (ADEM), Addison's disease, Ankylosing spondylitis, Antiphospholipid antibody syndrome (APS), Aplastic anemia, Autoimmune hepatitis, Autoimmune Oophoritis, Celiac disease, Crohn's disease, Diabetes mellitus type 1, Gestational pemphigoid, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, Idiopathic thrombocytopenic purpura, Kawasaki's Disease, Lupus erythematosus, Multiple sclerosis, Myasthenia gravis, Opsoclonus myoclonus syndrome (OMS), Optic neuritis, Ord's thyroiditis, Pemphigus, Pernicious anaemia, Polyarthritis in dogs, Primary biliary cirrhosis, Rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, Takayasu's arteritis, Temporal arteritis (also known as "giant cell arteritis"), Warm autoimmune hemolytic anemia, Wegener's granulomatosis.

[0024] "Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient's life or life expectancy. In some embodiments, the disease condition is cancer. In some embodiments, the disease condition is an autoimmune disease.

[0025] "B-cell associated diseases" as used herein include B-cell immunodeficiencies, autoimmune diseases and/or excessive/uncontrolled cell proliferation associated with B-cells (including lymphomas and/or leukemias). Examples of such diseases, wherein bispecific CARs of the invention may be used for therapeutic approaches include but are not limited to systemic lupus erythematosus (SLE), diabetes, rheumatoid arthritis (RA), reactive arthritis, multiple sclerosis (MS), pemphigus vulgaris, celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X-linked agammaglobulinaemis, pre-B acute lymphoblastic leukemia, systemic lupus erythematosus, common variable immunodeficiency, chronic lymphocytic leukemia, diseases associated with selective IgA deficiency and/or IgG subclass deficiency, B lineage lymphomas (Hodgkin's lymphoma and/or non-Hodgkin's lymphoma), immunodeficiency with thymoma, transient hypogammaglobulinaemia and/or hyper IgM syndrome, as well as virally-mediated B-cell diseases such as EBV mediated lymphoproliferative disease, and chronic infections in which B-cells participate in the pathophysiology.

[0026] "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to B-cell lymphomas (Hodgkin's lymphomas and/or non- Hodgkins lymphomas), brain cancer, breast cancer, colon cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer.

[0027] "Chemotherapeutic drugs" or "chemotherapeutic agents" as used herein refer to drugs used to treat cancer including but not limited to Albumin-bound paclitaxel (nab-paclitaxel), Actinomycin, Alitretinoin, All-trans retinoic acid, Azacitidine, Azathioprine, Bevacizumab, Bexatotene, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cetuximab, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Fluorouracil, Gefitinib, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Ipilimumab, Irinotecan, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizumab, Ofatumumab, Oxaliplatin, Paclitaxel, Panitumab, Pemetrexed, Rituximab, Tafluposide, Teniposide, Tioguanine, Topotecan, Tretinoin, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vorinostat, Romidepsin, 5 -fluorouracil (5-FU), 6-mercaptopurine (6- MP), Cladribine, Clofarabine, Floxuridine, Fludarabine, Pentostatin, Mitomycin, ixabepilone, Estramustine, or a combination thereof.

[0028] "Immune cell" as used herein refers to the cells of the mammalian immune system including but not limited to antigen presenting cells, B-cells, basophils, cytotoxic T-celis, dendritic cells, eosinophils, granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells and T-cells. [0029] "Immune response" as used herein refers to immunities including but not limited to innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity and/or overactive immunity.

[0030] "Subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. In some embodiments, the subject has cancer. In some embodiments, the subject had cancer at some point in the subject's lifetime. In various embodiments, the subject's cancer is in remission, is re-current or is non-recurrent.

[0031] "Mammal" as used herein refers to any member of the class Mammalia, including, without limitation, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

[0032] "Treatment" and "treating," as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented. Examples of cancer treatment include, but are not limited to, active surveillance, observation, surgical intervention, chemotherapy, immunotherapy, radiation therapy (such as external beam radiation, stereotactic radiosurgery (gamma knife), and fractionated stereotactic radiotherapy (FS )), focal therapy, systemic therapy, vaccine therapies, viral therapies, molecular targeted therapies, or a combination thereof. [0033] "Tumor," as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0034] "Therapeutic agents" as used herein refers to agents that are used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of developing, slow the progression of and/or cure, a disease. Diseases targeted by the therapeutic agents include but are not limited to carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematologic diseases, autoimmune diseases, and/or inflammatory diseases.

[0035] "Antibody" as used herein refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as Fab, F(ab')2, Fv, and other fragments which retain the antigen binding function of the parent antibody. In an embodiment, the antibody is an abzyme, having for example, sialidase activity. The abzymes may be polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as Fab, F(ab')2, Fv, and other fragments which retain the sialidase activity of the parent antibody.

[0036] "Monoclonal antibody" as used herein refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(ab')2, Fv, and others which retain the antigen binding function of the antibody. Monoclonal antibodies of any mammalian species can be used in this invention. In practice, however, the antibodies will typically be of rabbit or murine origin because of the availability of rabbit or murine cell lines for use in making the required hybrid cell lines or hybridomas to produce monoclonal antibodies.

[0037] "Humanized antibodies" as used herein means that at least a portion of the framework regions of an immunoglobulin are derived from human immunoglobulin sequences. [0038] "Single chain antibodies" as used herein refer to antibodies prepared by determining the binding domains (both heavy and light chains) of a binding antibody, and supplying a linking moiety which permits preservation of the binding function. This forms, in essence, a radically abbreviated antibody, having only that part of the variable domain necessary for binding to the antigen. Determination and construction of single chain antibodies are described in U.S. Pat. No. 4,946,778 to Ladner et al.

Production of catalytically active antibodies (abzymes)

[0039] Provided herein are abzymes or fragments thereof or derivative thereof, having sialidase activity. In an embodiment, the abzymes of the invention are obtained by using an inhibitor of neuraminidase as an antigen to produce the catalytic antibodies (abzymes) having sialidase activity. In some embodiments, the inhibitor of neuraminidase is an inhibitor of Neul, Neu2, Neu3 or Neu4. In an embodiment, the neuraminidase inhibitor binds to the active site of the neuraminidase. In some embodiments, the neuraminidase inhibitor is a small molecule. In an embodiment, the neuraminidase inhibitor is a hapten. In specific embodiments, the neuraminidase inhibitor may be any one or more of zanamivir, oseltamivir, siastatin B, laninamivir, peramivir, 2,3 -deny dro-2-deoxy-N-acetylneuraminic acid (DANA) or a variant thereof or a combination thereof. Other sialidase inhibitors will be apparent to one skilled in the art [Albohy A, et al. Identification of Selective Nanomolar Inhibitors of the Human Neuraminidase, NEU4. ACS Medicinal Chemistry Letters 2013; 4:532-537; Zhang Y, et al. Identification of Selective Inhibitors for Human Neuraminidase Isoenzymes Using C4,C7-Modified 2-Deoxy-2,3-didehydro-N-acetylneuraminic Acid (DANA) Analogues. Journal of Medicinal Chemistry 2013; 56:2948-2958; Tsai C-S, et al. Cell-permeable probe for identification and imaging of sialidases. Proceedings of the National Academy of Sciences 2013; 110:2466-2471 ; Magesh S, et al. Design, synthesis, and biological evaluation of human sialidase inhibitors. Part 1 : Selective inhibitors of lysosomal sialidase (NEU1). Bioorganic & Medicinal Chemistry Letters 2008; 18:532-537; Magesh S. et al., Human sialidase inhibitors: Design, synthesis, and biological evaluation of 4- acetamido-5-acylamido-2-fluoro benzoic acids. Bioorg Med Chem 2009; Magesh S, et al., Homology modeling of human sialidase enzymes NEU1, NEU3 and NEU4 based on the crystal structure of NEU2: hints for the design of selective NEU3 inhibitors. J Mol Graph Model 2006; 25: 196-207; Albohy A, et al. Insight into substrate recognition and catalysis by the human neuraminidase 3 (NEU3) through molecular modeling and site-directed mutagenesis. Glycobiology 2010; 20: 1 127-1 138].

[0040] In various embodiments, to prepare the antigen to produce the catalytically-active antibodies having sialidase activity, the antigen, namely the neuraminidase inhibitor (for example, the hapten DANA) is conjugated to a carrier molecule via a bridging group.

[0041] In some embodiments, the neuraminidase inhibitor comprises the bridging group. In some embodiments the bridging group is a naturally occurring part of the neuraminidase inhibitor and in other embodiments the neuraminidase inhibitors may be modified to attach a bridging group while still retaining its neuraminidase inhibition function. Example of bridging groups include but are not limited to any one or more of a carboxyl group, an amino group, an aldehyde group, a sulfhydryl group, a disulfide group, a hydroxyl group, or a combination thereof. The conjugation of the neuraminidase inhibitor to the carrier molecule may be facilitated by cross-linkers such as zero-length crosshnkers, for example l-Ethyl-3- [3-dimethylaminopropyl]carbodiimide hydrochloride (EDC or ED AC), dicyclohexyl carbodiimide (DCC); homobifunctional crosshnkers, for example N-hydroxysuccinimide (NHS) esters, 3,3'-dithiobis(succinimidylpropionate) (Lomant's reagent, DSP); heterobifunctional crosshnkers, for example N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP); dendrimers; other crosslinking agents will be apparent to one skilled in the art [Hermanson GT. Bioconjugate Techniques. San Diego, CA, USA: Academic Press; 785p, 1996].

[0042] In various embodiments, the carrier is any one or more of polyvinylpyrrolidone (PVP), bovine gamma-globulin (BGG), isologous mouse immunoglobulin (MIG), albumin, prealbumin, ox serum albumin, Megathura crenulata hemocyanin, Concholepas concholepas hemocyanin (CCH), Blue Carrier Protein, polyethylene glycol, polyacrylamide, agarose, purified protein derivative (PPD), KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), Cationized BSA, OVA (Ovalbumin), gelatin, multiple antigen peptides (MAPs), synthetic or natural polymeric matrices or combinations thereof.

[0043] In various embodiments, the antigen to produce the catalytically-active antibodies (abzymes) having sialidase activity comprises a neuraminidase inhibitor bearing a bridging group conjugated to a carrier. In an embodiment, the antigen to produce the catalytically- active antibodies having sialidase activity comprises the neuraminidase inhibitor DANA bearing a carboxyl bridging group conjugated to the carrier ox serum albumin. In an embodiment, the antigen to produce the catalytically-active antibodies having sialidase activity comprises the neuraminidase inhibitor DANA bearing a carboxyl bridging group conjugated to the carrier Megathura crenulata (keyhole limpet) hemocyanin (KLH) or bovine serum albumin (BSA).

[0044] Catalytically active antibodies using the antigens described herein may be produced by any one of several methods known in the art. For example, see Yoshida et al., Experientia 43:329, 1987; Yoshida and Ichiman, J Clin. Microbiol. 20:461, 1984; and U.S. Pat. No. 5,770,208 D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor NY, 1988); Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511 ; Queen et al. U. S. Patent No. 5,585,089; Riechmann et al, Nature 332: 323 (1988); and Ausebel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., N.Y., 1989. Classically, antigen-specific antibodies are produced by immunizing a host animal with the antigen (for example, the antigens set forth herein) and later collecting the antibody-containing serum from the animal.

[0045] Any animal capable of producing antibodies in response to an antigen may be used in the invention. Commonly used animals include: mice, rats, horses, cows, goats, sheep, rabbits, cats, dogs, guinea pigs, chickens and humans. Host animals are immunized by injection with the antigen (for example, DANA conjugated to a carrier protein). Preferably, after the first immunization, the host animal receives one or more booster injections of antigen to augment antibody production and affinity. For immunization of humans, care should be taken to select the appropriate antigen, adjuvant, and/or carrier protein to avoid potential adverse reactions (e.g., granuloma formation with Freund's complete adjuvant; anaphylactic shock).

[0046] To enhance the immunologic response antigens are typically mixed with adjuvant before injecting into a host animal or human. Adjuvants useful in augmenting antibody production include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol (DNP). Examples of potentially useful human adjuvants include BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Antigens can also be cross-linked or incorporated into lipid vesicles to enhance their antigenicity.

[0047] Antibodies within the invention include without limitation polyclonal antibodies, monoclonal antibodies, humanized, and chimeric antibodies. Polyclonal antibodies can be isolated by collecting sera from immunized host animals. Monoclonal antibodies can be prepared using the antigens discussed above and standard hybridoma technology. See, e.g., Kohler et al., Nature, 256:495, 1975; Kohler et al, Eur. J. Immunol, 6:51 1, 1976; Kohler et al., Eur. J. Immunol, 6:292, 1976; Hammerling et al., "Monoclonal Antibodies and T cell Hybridomas," Elsevier, N.Y., 1981 ; Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y., 1997. Human monoclonal antibodies are prepared by immortalizing a human antibody secreting cell (e.g., a human plasma cell). See, e.g., U.S. Pat. No. 4,634,664. To obtain monoclonal antibodies, hybridomas or other immortalized antibody secreting cells are cultivated in vitro (e.g., in tissue culture) or in vivo (e.g., in athymic or SCID mice). Antibodies are isolated by collecting the in vitro culture medium or bodily fluids (e.g., serum or ascites) from the in vivo cultures.

[0048] Additionally, chimeric antibodies, which are antigen-binding molecules having different portions derived from different animal species (e.g., variable region of a rat immunoglobulin fused to the constant region of a human immunoglobulin), are expected to be useful in the invention. Such chimeric antibodies can be prepared by methods known in the art. E.g., Morrison et al, Proc. Nat'l. Acad. Sci. USA, 81 :6851, 1984; Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452, 1984. Similarly, antibodies can be humanized by methods known in the art. For example, monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, Calif.) or as described in U.S. Pat. Nos. 5,693,762; 5,530,101 ; or 5,585,089. In a like manner, portions of the constant region of Protein A- or Protein G-binding immunoglobulins can be altered, deleted or mutated to remove Protein A or Protein G reactivity.

[0049] Once isolated, antibodies can be further purified by conventional techniques including: salt cuts (e.g., saturated ammonium sulfate precipitation), cold alcohol fractionation (e.g., the Cohn-Oncley cold alcohol fractionation process), size exclusion chromatography, ion exchange chromatography, immunoaffinity chromatography (e.g., chromatography beads coupled to anti-human immunoglobulin antibodies can be used to isolate human immunoglobulins) and antigen affinity chromatography. See, e.g., Coligan et al., supra. Conventional antibody purification techniques using Protein A and Protein G (e.g., Protein A or Protein G chromatography) may be utilized.

[0050] Standard techniques in immunology and protein chemistry can be used to analyze and manipulate the antibodies of the invention. For example, dialysis can be used to alter the medium in which the antibodies are dissolved. The antibodies may also be lyophilized for preservation. Antibodies can be tested for their sialidase activity as described herein. Antibodies can be tested for the ability to bind specific antigens using any one of several standard methods such as Western Blot, immunoprecipitation analysis, enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). See, e.g., Coligan et al., supra.

[0051] Monoclonal antibodies may be prepared using the method of Kohler and Milstein, Nature (1975) 256:495-96, or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) are- removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT"). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the desired immunizing cell-surface antigen (and which do not bind to unrelated antigens) and have sialidase activity. The selected mAb-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

[0052] If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32P and 1251), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3',5,5'- tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. "Specific binding partner" refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, ¹²⁵I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a mAb. Further, one may combine various labels for desired effect. For example, mAbs and avidin also require labels in the practice of this invention: thus, one might label a mAb with biotin, and detect its presence with avidin labeled with ¹²⁵I, or with an anti-biotin mAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.

[0053] In various embodiments, the abzyme produced by the methods described herein, having sialidase activity, is a monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, chimeric antibodies, humanized antibodies, human antibodies, or a single chain antibody. In various embodiments, the fragments of the abzymes produced by the methods described herein are obtained by treating the abzymes with proteases. For example, treating the abzyme of the invention with papain may yield three fragments, namely two Fab fragments and one Fc fragment. Alternately, treating the abzyme of the invention with pepsin may yield two fragments, namely F(ab')₂ and Fc. In some embodiments, the fragments of the abzymes are also catalytically.

Therapeutic uses of the catalytically active antibodies (abzymes)

[0054] The invention provides a method for treating autoimmune disease and/or cancer in a subject in need thereof. The method includes providing a composition comprising an abzyme having sialidase activity and administering an effective amount of the composition to the subject so as to treat autoimmune disease and/or cancer in the subject. In some embodiments, the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof. In one embodiment, the targeting element is fused to the abzyme or a fragment thereof. In another embodiment, the targeting element is conjugated to the abzyme or a fragment thereof. In various embodiments, the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof, that recognizes a specific marker on the surface of apoptotic cells and/or cancer cells. In some embodiments, the compositions comprising an abzyme having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen) and a targeting element may be administered in conjunction with other therapeutic agents used in autoimmune diseases therapy.

[0055] The invention further provides methods for inhibiting autoimmune disease and/or cancer in a subject in need thereof. The method includes providing a composition comprising an abzyme having sialidase activity and administering an effective amount of the composition to the subject so as to inhibit autoimmune and/or cancer disease in the subject. In some embodiments, the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof. In one embodiment, the targeting element is fused to the abzyme or a fragment thereof. In another embodiment, the targeting element is conjugated to the abzyme or a fragment thereof. In various embodiments, the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof, that recognizes a specific marker on the surface of apoptotic cells and/or cancer cells. In some embodiments, the compositions comprising an abzyme having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen) and a targeting element may be administered in conjunction with other therapeutic agents used in autoimmune diseases therapy.

[0056] The invention further provides methods for reducing the symptoms of autoimmune disease and/or cancer in a subject in need thereof. The method includes providing a composition comprising an abzyme having sialidase activity and administering an effective amount of the composition to the subject so as to reduce the symptoms of autoimmune and/or cancer disease in the subject. In some embodiments, the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof. In one embodiment, the targeting element is fused to the abzyme or a fragment thereof. In another embodiment, the targeting element is conjugated to the abzyme or a fragment thereof. In various embodiments, the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof, that recognizes a specific marker on the surface of apoptotic cells and/or cancer cells. In some embodiments, the compositions comprising an abzyme having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen) and a targeting element may be administered in conjunction with other therapeutic agents used in autoimmune diseases therapy.

[0057] The invention further provides methods for promoting prophylaxis of autoimmune disease and/or cancer in a subject in need thereof. The method includes providing a composition comprising an abzyme having sialidase activity and administering an effective amount of the composition to the subject so as to promote prophylaxis of autoimmune and/or cancer disease in the subject. In some embodiments, the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof. In one embodiment, the targeting element is fused to the abzyme or a fragment thereof. In another embodiment, the targeting element is conjugated to the abzyme or a fragment thereof. In various embodiments, the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof, that recognizes a specific marker on the surface of apoptotic cells and/or cancer cells. In some embodiments, the compositions comprising an abzyme having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen) and a targeting element may be administered in conjunction with other therapeutic agents used in autoimmune diseases therapy.

[0058] The invention further provides methods for increasing clearance of apoptotic cell in a subject in need thereof. The method includes providing a composition comprising an abzyme having sialidase activity and administering an effective amount of the composition to the subject so as to increase clearance of apoptotic cell in the subject. In some embodiments, the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof. In one embodiment, the targeting element is fused to the abzyme or a fragment thereof. In another embodiment, the targeting element is conjugated to the abzyme or a fragment thereof. In various embodiments, the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof, that recognizes a specific marker on the surface of apoptotic cells and/or cancer cells.

[0059] The invention further provides methods treating, inhibiting, reducing the severity of and/or promoting prophylaxis of a disease-state resulting from reduced clearance of apoptotic cells in a subject in need thereof. The method includes providing a composition comprising an abzyme having sialidase activity and administering an effective amount of the composition to the subject so as to treat, inhibit, reduce the severity of and/or promote prophylaxis of a disease-state resulting from reduced clearance of apoptotic cells in the subject. In some embodiments, the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof. In one embodiment, the targeting element is fused to the abzyme or a fragment thereof. In another embodiment, the targeting element is conjugated to the abzyme or a fragment thereof. In various embodiments, the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof, that recognizes a specific marker on the surface of apoptotic cells and/or cancer cells. In some embodiments, the compositions comprising an abzyme having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen) and a targeting element may be administered in conjunction with other therapeutic agents used in the disease-state.

[0060] The invention further provides methods treating, inhibiting, reducing the symptoms of and/or promoting prophylaxis of cancer in a subject in need thereof. The method includes providing a composition comprising an abzyme having sialidase activity and administering an effective amount of the composition to the subject so as to treat, inhibit, reduce the severity of and/or promote prophylaxis of cancer in the subject. In some embodiments, the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof. In one embodiment, the targeting element is fused to the abzyme or a fragment thereof. In another embodiment, the targeting element is conjugated to the abzyme or a fragment thereof. In various embodiments, the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof, that recognizes a specific marker on the surface of apoptotic cells and/or cancer cells. In some embodiments, the compositions comprising an abzyme having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen) and a targeting element may be administered in conjunction with other therapeutic agents used in cancer therapy.

[0061] The invention further provides methods treating, inhibiting, reducing the symptoms of and/or promoting prophylaxis of B-cell associated diseases in a subject in need thereof. The method includes providing a composition comprising an abzyme having sialidase activity and administering an effective amount of the composition to the subject so as to treat, inhibit, reduce the severity of and/or promote prophylaxis of B-cell associated diseases in the subject. In some embodiments, the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof. In one embodiment, the targeting element is fused to the abzyme or a fragment thereof. In another embodiment, the targeting element is conjugated to the abzyme or a fragment thereof. In various embodiments, the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof, that recognizes a specific marker on the surface of apoptotic cells and/or cancer cells. In an embodiment, the B-cell associated disease is SLE. In some embodiments, the compositions comprising an abzyme having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen) and a targeting element may be administered in conjunction with other therapeutic agents used in therapy of B-cell associated diseases.

[0062] In various embodiments, the abzyme in the compositions for therapeutic uses is produced by the methods described herein. In an embodiment, the antigen used to produce the catalytically-active antibodies (abzyme) having sialidase activity for the therapeutic uses described herein comprises the neuraminidase inhibitor DANA bearing a carboxyl bridging group conjugated to the carrier bovine serum albumin. In an embodiment, the antigen to produce the catalytically-active antibodies (abzyme) having sialidase activity for the therapeutic uses described herein comprises the neuraminidase inhibitor DANA bearing a carboxyl bridging group conjugated to the carrier Megathura crenulata hemocyanin. In various embodiments, the abzyme selected from the group consisting of monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, chimeric antibodies, humanized antibodies, human antibodies and a single chain antibody. Markers of Apoptotic Cells

[0063] In various embodiments, the composition for the therapeutic uses described herein comprises the catalytically-active antibodies having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen, as described herein) and a targeting element that targets markers on the surface of apoptotic cells. Examples of the targets include but are not limited to any one or more of negatively charged lipids, phosphatidylserine (PS), alpha- D-mannose-specific plasma membrane glycoepitopes, beta-D-galactose-specific plasma membrane glycoepitopes, Annexin I, calreticulin, nucleolin, PARP-1, APO-1 , or a combination thereof.

[0064] Surface markers on apoptotic cells are necessary for apoptotic cell recognition and subsequent clearance by cells of the immune system. Apoptosis is accompanied by the loss of plasma membrane symmetry. Characteristic of this change is the exposure of amine- containing phospholipids such phosphatidylserine (PS) to cell surfaces. This exposure is functionally important as it provides a signal for recognition and elimination of apoptotic cells by macrophages and phagocytes. Such a change in cell membrane properties allows identification and characterization of apoptotic cells (see US Patent No.7880021).

[0065] Further, cell surface glycoconjugates also change on apoptotic cells. For example, alpha-D-mannose and beta-D-galactose-specific plasma membrane glycoepitopes have substantially increased expression after induction of apoptosis (Bilyy and Stoika, Search for novel cell surface markers of apoptotic cells, Autoimmunity. 2007 Jun 40(4):249- 53). Additionally, Annexin I and calreticulin are also exposed on the cell surface during apoptotic cell clearance (Elmore, Apoptosis: A Review of Programmed Cell Death, Toxicol Pathol. 2007; 35(4): 495-516). Other changes on the cell surface of apoptotic cells include the expression of thrombospondin binding sites, loss of sialic acid residues, nucleolin mislocalized to the plasma membrane, PARPl-1, and APO-1 (Zhang et al., Early Detection of Apoptosis Using a Fluorescent Conjugate of Annexin V, Biotechniques. 1997 Sep;23(3):525-31 ; Bates et al, A method for the detection of apoptosis, WO 2004003554 Al ; Krammer, A cell surface antigen associated with cellular apoptosis, WO 1991010448 Al). Other markers for apoptotic cells that may be targeted by the targeting elements will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention [Bilyy RO, et al. Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles. J Biol Chem 2012; 287:496-503; Stoika RS, et al. Agglutination-based method for fast detection, isolation and quantification of apoptotic cells. USA, UA patent WO/2007/053654; Fadok VA, et al., Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol. 1992; 148:2207-2216; Bilyy R, Stoika R. Search for novel cell surface markers of apoptotic cells. Autoimmunity 2007; 40:249-253; Hochreiter- Hufford A, Ravichandran KS. Clearing the Dead: Apoptotic Cell Sensing, Recognition, Engulfment, and Digestion. Cold Spring Harbor Perspectives in Biology 2013; 5; Li W. Eat- me signals: Keys to molecular phagocyte biology and "Appetite" control. Journal of Cellular Physiology 2012; 227: 1291-1297].

[0066] In various embodiments, administering a composition comprising the catalytically- active antibodies having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen) and a targeting element that targets apoptotic cells allows desialylation of the target apoptotic cells while leaving the healthy cells intact. For example, a composition comprising the sialidase abzyme produced by the method described herein and an antibody that targets annexin I, may result in apoptotic cells being targeted (due to the annexin I antibody) and desialylated (due to the sialidase abzyme), thus facilitating clearance of apoptotic cells.

Markers of cancer cells

[0067] In various embodiments of the methods described herein, the composition for the therapeutic uses comprises the catalytically-active antibodies having sialidase activity (for example, produced using a neuraminidase inhibitor as an antigen) and a targeting element targets markers on the surface of cancer cells. Markers on the surface of cancer cells that may be targeted by the targeting elements of the compositions described herein include but are not limited to 4- IBB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B- lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221 , CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EGFRVIII, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, FIGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, LI -CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or vimentin. Other antigens specific for cancer will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0068] In various embodiments, administering a composition comprising the sialidase abzyme and a targeting element that targets cancer cells allows desialylation of the target cancer cells while leaving the healthy cells intact, thus marking the cancer cells for clearance or further facilitating clearance of the target cancer cells. For example, a composition comprising the sialidase abzyme produced by the method described herein and an antibody that targets EGFRVIII, may result in, for example, glioblastoma cells being targeted (due to the EGFRVIII antibody) and desialilyted (due to the sialidase abzyme), thus marking the cells for apoptotic clearance and/or facilitating clearance of cancerous apoptotic cells

Agents and Elements Targeting Cells

[0069] Various agents and elements capable of recognizing and binding to surface markers on apoptotic cells and/or cancer cells can be used to target the sialidase abzyme to apoptotic cells and/or cancer cells. Examples of these agents and elements include, but are not limited to, (1) antibodies and peptides recognizing apoptotic surface markers, such as negatively charged lipids, phosphatidylserine (PS), alpha-D-mannose-specific plasma membrane glycoproteins, beta-D-galactose-specific plasma membrane glycoproteins, Annexin I, calreticulin, Nucleolin, PARP-1, and APO-1 ; (2) Annexin V and a variant thereof; (3) thrombospondin and a variant thereof for recognizing and binding to thrombospondin binding sites on apoptotic cells; (4) antibodies and peptides for recognizing and binding to cancer markers; (5) compounds such as 3-hydroxyflavone derivatives, TCN12S, F2N12S, and an organic molecule composed of a fluorophore and an artificial zinc-containing receptor for PS recognition; and (6) oligonucleotides that bind to Nucleolin. (See Demchenko et. AL, Compounds and kits for the detection and the quantification of cell apoptosis, US 7880021; Bates et al., A method for the detection of apoptosis, WO 2004003554 Al). The targeting agent or element can be fused with Abzyme to form a fusion protein or can be conjugated/ linked/complexed to abzyme. If the targeting agent or element is an antibody, a bi-specific antibody can be formed to comprise a targeting antibody and the abzyme.

PHARMACEUTICAL COMPOSITIONS

[0070] In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of the catalytically active antibody having sialidase activity produced by the methods described herein, so as to treat, inhibit, reduce the symptoms of and/or promote prophylaxis of autoimmune diseases and/or cancer. In some embodiments, the pharmaceutical composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof. In one embodiment, the targeting element is fused to the abzyme or a fragment thereof. In another embodiment, the targeting element is conjugated to the abzyme or a fragment thereof. In some embodiments, the apoptotic cell markers and the cancer cell markers that may be targeted by the targeting element of the pharmaceutical composition are set forth herein. In various embodiments, the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof, that recognizes a specific marker on the surface of apoptotic cells and/or cancer cells.

[0071] "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0072] In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal, parenteral or enteral. "Parenteral" refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Typically, the antibodies are administered by injection, either intravenously or intraperitoneally. Methods for these administrations are known to one skilled in the art.

[0073] The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0074] The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0075] The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0076] The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0077] Before administration to patients, formulants may be added to the antibodies (such as the abzymes having sialidase activity produced by the methods described herein). A liquid formulation may be preferred. For example, these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, bulking agents or combinations thereof.

[0078] Carbohydrate formulants include sugar or sugar alcohols such as monosaccharides, disaccharides, or polysaccharides, or water soluble glucans. The saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof. "Sugar alcohol" is defined as a C₄ to C₈ hydrocarbon having an —OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. In one embodiment, the sugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %, more preferable between 2.0 and 6.0 w/v %.

[0079] Amino acids formulants include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added.

[0080] In some embodiments, polymers as formulants include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000.

[0081] It is also preferred to use a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution. Most any physiological buffer may be used including but not limited to citrate, phosphate, succinate, and glutamate buffers or mixtures thereof. In some embodiments, the concentration is from 0.01 to 0.3 molar. Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268,1 10.

[0082] Additionally, antibodies (such as the abzymes having sialidase activity produced by the methods described herein) can be chemically modified by covalent conjugation to a polymer to increase their circulating half-life, for example. Preferred polymers, and methods to attach them to peptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546 which are all hereby incorporated by reference in their entireties. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and in some embodiments, has an average molecular weight between 1000 and 40,000, between 2000 and 20,000, or between 3,000 and 12,000. In some embodiments, PEG has at least one hydroxy group, such as a terminal hydroxy group. The hydroxy group may be activated to react with a free amino group on the inhibitor. However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/antibody of the present invention.

[0083] Water soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. POG is preferred. One reason is because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body. The POG has a molecular weight in the same range as PEG. The structure for POG is shown in Knauf et al., 1988, J. Bio. Chem. 263: 15064-15070 and a discussion of POG/IL C 2 conjugates is found in U.S. Pat. No. 4,766,106, both of which are hereby incorporated by reference in their entireties.

[0084] Another drug delivery system for increasing circulatory half-life is the liposome. Methods of preparing liposome delivery systems are discussed in Gabizon et al., Cancer Research (1982) 42:4734; Cafiso, Biochem Biophys Acta (1981) 649: 129; and Szoka, Ann Rev Biophys Eng (1980) 9:467. Other drug delivery systems are known in the art and are described in, e.g., Poznansky et al., Drug Delivery Systems (R. L. Juliano, ed., Oxford, N.Y. 1980), pp. 253-315; M. L. Poznansky, Pharm Revs (1984) 36:277.

[0085] After the liquid pharmaceutical composition is prepared, it may be lyophilized to prevent degradation and to preserve sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is administered to subjects using those methods that are known to those skilled in the art.

[0086] The dosage and mode of administration will depend on the individual. Generally, the compositions are administered so that antibodies are given at a dose between ^g/kg and 20 mg/kg, between 20μg/kg and 10 mg/kg, between lmg/kg and 7 mg/kg. In some embodiments, it is given as a bolus dose, to increase circulating levels by 10-20 fold and for 4-6 hours after the bolus dose. Continuous infusion may also be used after the bolus dose. If so, the antibodies may be infused at a dose between 5μg/kg/minute and 20μg/kg/minute, or between 7μg/kg/minute and 15μg/kg/minute.

KITS OF THE INVENTION

[0087] The invention also provides a kit to treat, inhibit, reduce the symptoms of and/or promote prophylaxis of autoimmune diseases and/or cancer in a subject in need thereof. The kit comprises a composition comprising the catalytically active antibody having sialidase activity produced by the methods described herein and instructions for use of the composition for treating, inhibiting and/or reducing the severity of autoimmune diseases and/or cancer in subjects in need thereof.

[0088] The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including the catalytically active antibody having sialidase activity produced by the methods described herein, as described above.

[0089] The exact nature of the components configured in the inventive kit depends on its intended purpose. In one embodiment, the kit is configured particularly for human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.

[0090] Instructions for use may be included in the kit. "Instructions for use" typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as so treat, inhibit, reduce the symptoms of and/or promote prophylaxis of autoimmune diseases and/or cancer in a subject. Optionally, the kit also contains other useful components, such as, measuring tools, diluents, buffers, pharmaceutically acceptable carriers, syringes or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0091] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a bottle used to contain suitable quantities of an inventive composition containing the catalytically active antibody having sialidase activity produced by the methods described herein. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components. EXAMPLES

[0092] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1

Experimental Methods

Patients

[0093] Peripheral blood serum samples of 65 patients diagnosed with SLE (with SLEDAI>4 [Gladman DD, et ah Systemic lupus erythematosus disease activity index 2000. The Journal of Rheumatology 2002; 29:288-91]) were analyzed. An informed consent was obtained from all patients, as it was approved by the Review Board of the Lviv National Medical University, in accordance with the regulations of the Ministry of Health Protection of Ukraine.

Animals

[0094] 8 rabbits were used - 2 were immunized with DANA-BSA, 2 with BSA, 2 with DANA-KLH and 2 with KLH. Immunization was performed according to [Kit Y, et ah Immunogenicity and Adjuvant Properties of Novel Biocompatible Nanoparticles. In: Kumar A, ed. Biocompatible Nanomaterials: Synthesis, Characterization and Applications. New York: Nova Publisher, 2010:209-23] using a mixture of DANA-protein conjugates (20μg/injection) synthesized as herein, and Freund adjuvant (50μ1/ΐη)εϋίϊοη) in PBS (total volume 400μ1) (complete Freund adjuvant was used during 1 st immunization, and incomplete Freund adjuvant at 2nd and 3rd immunizations; 4th immunization was with protein adjuvants alone, blood was collected 10 days after 4th immunization. Food and water were available ad libitum and all experiments with animals were approved by the Bioethics Committee of Institute of Cell Biology, NAS of Ukraine. Cell culture and isolation of cells

[0095] Human leukemia Jurkat T-cells, as well as primary human PMN and MoMa from healthy volunteers was used. Monocytes were isolated from peripheral blood by LymphoPrep^® gradient according to manufacturer's recommendations for isolation of PBMC fraction. Plastic-attached cells of PBMC fraction were then cultured for 7 days in the presence of GM-CSF (l OOU/ml) and autologous serum (added at days 1, 3 and 5) to generate MoMa. After 7 days of differentiation, the MoMa population was tested. They typically contain >95% CDl lb+ cells, >90% CD14+ cells and >85% CD89+ cells. Phagocytosis was assessed by incubation of PMN (freshly isolated or aged for 24 h) with sialyl abzyme, its F(ab)₂ fragment, or C. perfringens neuraminidase (each at normalized activity of 30mU) for 3 h at 37°C at Ringer buffer. Cells were thoroughly washed three times with Ringer solution and incubated with human MoMa. Uningested PMN were analyzed by flow cytometry (for this reason cells were pre-stained with CFSE [Rodel F, et al. The CFSE distribution assay is a powerful technique for the analysis of radiation-induced cell death and survival on a single- cell level. Strahlentherapie und Onkologie: Organ der Deutschen Rontgengesellschaft [et al] 2005; 181 :456-62] or in the hemocytometric chamber using Zeiss Axiolmager Al microscope. The percentage of prey cells that had been bound to or taken up by MoMa was calculated.

Induction and inhibition of apoptosis

[0096] Cell viability was controlled by Annexin V/PI staining. Apoptosis was induced by irradiation of Jurkat cells with ultraviolet light type B (UV-B) (180 mJ/cm2, 60 s), or by ageing of polymorphonuclear leukocytes (PMN).

Flow cytometry

[0097] Analyses employing fluorescence-labeled lectins [Franz S, et al. Lectins detect changes of the glycosylation status of plasma membrane constituents during late apoptosis. Cytometry A 2006; 69:230-9] were performed using FACS Scan flow cytometer (BD Biosciences). PI was used to counter-stain necrotic cells, which were excluded from analysis. Lectin PNA (peanut agglutinin) and SNA (Sambucus nigra agglutinin II, a2,6-sialil specific) were from Lectinotest Laboratory (Ukraine). Antibody purification

[0098] Isolation of IgG fractions from blood serum was performed according to the procedure reported [Bilyy R, et al. Cell surface glycans at SLE: changes during cells death, utilization for disease detection and molecular mechanism underlying their modification. In: Mavragani C, ed. Autoimmune Disorder I Book 1 : INTECH, 201 1 :89-110], the purification methods used in this study are summarized in Figure 2. Specifically, blood serum proteins were 3 times precipitated with ammonium sulphate (50% saturation), the pellet was dissolved in 150 mM NaCl, 20 mM Tris-HCl buffer, pH 7.5, and dialyzed against the same buffer. IgGs were purified by affinity chromatography employing Protein G-Sepharose column. IgG was eluted from the column with 0.1 M glycine-HCl, pH 2.6, immediately neutralized by 1 M Tris-HCl buffer, pH 8.8, and dialyzed for 18 h against 100 mM NaCl, 20 mM Tris-HCl buffer, pH 7.5. Protein concentration was measured by the NanoDrop ND 1000 spectrophotometer using extinction coefficient of IgG, preloaded in the device (NanoDrop Technologies, USA). The IgG-Ab were tested for sialidase activity.

HPLC

[0099] Size exclusion high performance liquid chromatography (HPLC-SEX) was performed in PBS, pH 6.8 on Perkin Elmer HPLC Series 200 HPLC system using Bio-Sil SEC 250 7.8*300 mm column (Bio-Rad) at 1 ml/min flow rate. The fractions corresponding to the main peak were collected and used for further analysis. Analysis of immune complexes was performed using the same setup in 0.1 M Glycin-HCl with 0.05% azide, pH 2.6 supplied at 1 ml/min flow rate.

[0100] Strong cation exchange high performance liquid chromatography (HPLC-SCX) was performed using Shiseido Capcell SCX UG80 1.5mm* 150mm column with 20mMES plus gradient of NaCl (from 60 to 200 mM) with a flow rate of 0.3 ml/min.

Preparation of F(ab)2 fragments

[0101] Antibodies were digested with pepsin, undigested antibodies were removed with protein-G-sepharose, F(ab)2 fragments were purified by gel filtration chromatography with the Toyopearl HW-55 F column (0.5x20 cm) in 50 mM sodium phosphate, 100 mM NaCl, Tween 20 0.25 M buffer, pH 7.0, 0.1 % NaN3 or by HPLC SEC using Bio-Sil SEC 250 column to remove potential contaminations with immune complexes.

Fluorometric assay

[0102] Sialidase activity was measured, as described [Warner TG, O'Brien JS. Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis. Biochemistry 1979; 18:2783-7; Tomin A, et al. Novel assay for direct fluorescent imaging of sialidase activity. Proc SPIE 2011 ; 8087:808769]. Briefly, protein samples (50 μg of protein per sample) were diluted to equal protein concentration (estimated by Bradford protein assay) with 0.2 M acetate buffer (with 5 mM CaC12 and MgC12), pH 4.2 in final volume of sample at 190 μΐ, 10 μΐ of 0.5 mM 4-MU- NA or 4-MU-Gal (both from BioSynth, Switzerland) were added and incubated for 3 h. In some cases 4-MU-Gal was used as substrate specificity control. If indicated, sialidase inhibitor DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid, Sigma, in final concentration 15 μΜ) was added to the protein samples. Cleavage of the substrate by sialidase yields the fluorescent product, 4-methylumbelliferone. Substrate used without Ab, served as a blank to determine the nonspecific degradation of the substrate. Reaction was stopped by the addition of 1 ml of glycine-carbonate buffer, pH 10.7 and fluorescence was measured (excitation - 365 nm, and emission - 430 nm) using a Turner Quantech FM109510 (USA) fiuorometer.

SDS-PAGE

[0103] Ab and their (Fab)2 fragments (3-5 μg per well) under the non-reducing or reducing (in the presence of 2% 13-mercaptoethanole, β-ΜΕ) conditions was performed in 5-16% gradient gels containing 0.1% SDS in Laemmli system.

Statistics

[0104] Statistical significance was assessed employing the Student's t-test. Three levels of significance were depicted with asterisks *- p<0.05; **- p<0.01 ; ***- pO.001. Example 2

Antibody Production

[0105] Typical methods for obtaining synthetic catalytically-active antibodies (abzymes) with sialidase activity includes immunizing animals (for example, rabbits) with haptens that imitate the transitional state of a catalyzed enzymatic reaction. The introduction of haptens (conjugated to carriers such as proteins) into the organism leads to the formation of a range of antibodies, among which are those which stabilize the compounds in the transitional state of the catalyzed reaction and in this way reduce the activation energy of the catalyzed reaction and ensuring that it proceeds. The following can serve as examples of obtaining induced abzymes.

[0106] An example of obtaining induced abzyme is a method of obtaining synthetic, catalytically-active antibodies capable of splitting phosphorus-bound compounds (sarin, for example) [International application WO/2003/039471 of 06 Nov 2002, IPC C07K 16/40 (Jan 2006), C12N 5/16 (Jan 2006), Catalytic Antibodies Raised Against Sarin And Process For The Preparation Thereof // publ. 15 May 2003]. This method is based on the synthesis of a hapten that would imitate the compounds of the transitional state of sarin splitting, by means of the conjugation of Cr(III) triene with N12,N6-di(0,0-diisopropyl)phosphoryl-L-lysine and its conjugation with a hemocyanin and by performing subsequent immunizations. A drawback of this method is its limitation with respect to the selection of suitable compound antigens that are capable of mimicking only phospho-organic compounds like the substrates of a catalyzed enzymatic reaction.

[0107] Another example of obtaining induced abzyme is a method of obtaining synthetic, catalytically-active antibodies capable of splitting cocaine [International application WO/1993/020076 of 02 Apr 1993, IPC A61K 47/48 (Jan 2006), C07N 451/02 (Jan 2006), C07F 9/6561 (Jan 2006), C07F 9/6571 (Jan 2006), C07F 9/6584 (Jan 2006), C07K 16/44 (Jan 2006), C12N 9/00 (Jan 2006), A61K 38/00 (Jan 2006), Catalytic Antibodies Against Cocaine And Methods Of Using And Producing Same // publ. 14 Oct 1993, Canadian patent CA2132982 of 26 Sep 1994, US patent US 08313291 of 03 Oct 1994, European patent EPO 1993910571 of 03 Nov 1994]. This method is based on the synthesis of a hapten that would imitate compounds of the transitional state of cocaine splitting cocaine, cocaine benzoyl esters, by means of multistage synthesis from a number of predecessors. The method foresees the immunization of animals with the haptens obtained or their conjugates with proteins and subsequent immunization of animals with the goal of obtaining catalytically- active antibodies to cocaine. The drawback to this method is its tendency to form only catalytically active antibodies capable of splitting cocaine and the complex, multistage scheme for the synthesis of the haptens.

[0108] A common drawback of these approaches to obtain induced abzymes is the original method of hapten synthesis directed toward inducing abzymes with the targeted catalytic activity (phospo-organic compounds or cocaine) and the absence of technical solutions for ensuring that catalytically-active antibodies with sialidase activity are induced or the presentation of unified approaches that could be used to create catalytically-active antibodies with sialidase activity.

[0109] According to the instant invention, a targeted development method is set forth obtaining catalytically-active antibodies with sialidase activity. Synthetic derivatives of a sialidase inhibitor are used as a hapten (an immunogenic agent) to obtain catalytically-active antibodies with sialidase activity for the immunization of rabbits, which inhibitor is conjugated with a high-molecular-weight carrier (for example, a protein molecule).

[0110] Accordingly, the inventors set forth a method for inducing catalytically-active antibodies with sialidase activity using derivatives of sialidase (neuraminidase) inhibitors as a hapten, which method is based on the conjugation of the inhibitor molecules, which contains a carboxyl or amide group, with a molecule of the protein carrier by means of the cross-linked reaction of zero length for the scheme depicted in Figure 1.

[0111] For inducing antibodies with catalytic sialidase activity, a sialidase inhibitor was used for the immunization. A protein carrier, BSA or KLH was dissolved in a concentration of 10 mg/ml in a reaction medium of 0.1 MES (2-(N- morpholino) ethansulfonic acid), pH 4.7. The sialidase inhibitor DANA was dissolved in 1M MES buffer, pH 4.7. The inhibitor solution was added to the protein solution in at least 10 times (molar) excess relative to the protein content. EDC was added to the reaction mixture in at least 10 times the molar excess relative to the protein content. The mixture was stirred until dissolved and incubated for two hours at 25°C. The conjugate was purified by gel filtration in a Sephadex G-50 or by dialysis, using a 0.01M solution of sodium phosphate and 0.15M sodium chloride, pH 7.4. The conjugated haptens obtained were used for the immunization of rabbits. The immunization of the rabbits was accomplished by mixing the conjugated DANA hapten (20μg/injection) and Freund's adjuvant (50μ1/ίη)εϋΐίοη) in a buffered physiological solution (BPS) with a total volume of 400μ1. For the immunization series, complete Friend's adjuvant was used in the first immunization, incomplete Friend's adjuvant for the second and third immunizations, and only the hapten solution in the fourth immunization. The time between immunizations was two weeks. Blood samples were taken 10 days after the fourth immunization, and separation of the antibodies was done as previously described [Kit et al., Immunogenicity and Adjuvant Properties of Novel Biocompatible Nanoparticles. Biocompatible Nanomaterials: Synthesis, Characterization and Applications, 2010. 209- 223].

[0112] For the immunization, four control animals and four animals that had been immunized with the herein described hapten-conjugate were used. If necessary, from the fraction of blood-serum antibodies, a fraction of class-IgG immunoglobulins were additionally purified, using affinity chromatography on protein-O-sepharose according to standard procedures [Bilyy, et al. Antibody-mediated sialidase activity in blood serum of patients with multiple myeloma. JMol Recognit, 2011 24:576-84].

[0113] The method proposed is differentiated from those described previously in that the method described herein it allows catalytically-active antibodies with sialidase activity to be obtained. In order to induce these antibodies, the conjugate of a derivative of a sialidase (neuraminidase) inhibitor is used as a hapten, for example DANA or another compound and a high-molecular- weight (protein) carrier.

Example 3

Antibody Purification

[0114] Two sources of abzymes with sialidase activity and their F(ab)₂ fragments were used in the instant application: 1) those isolated from sera of SLE patients after preliminary screening of a cohort of patients for sialidase activity in the globulin fraction of their sera; 2) those isolated using the methods described herein. [0115] The antibodies were purified as described above in Example 1. The IgG fractions obtained after ammonium sulphate precipitation and protein-G affinity chromatography were further subjected to HPLC SEC chromatography (the scheme of antibody purification is summarized in Figure 2) and the middle of the peaks corresponding to IgG were collected and used for analysis by SDS-PAGE, for testing neuraminidase activity and its inhibition with DANA (Figure 6). This type of purification (ammonium sulphate precipitation, Protein G-sepharose affinity chromatography, HPLC SEC separation) was previously reported to provide essentially pure IgG preparations containing abzymes with sialidase activity and lack immune complexes. The purity was confirmed by HPLC-SEC at pH 2,6, HPLC-SCX at the gradient of NaCl of 60-200mM (Figure 3) and by in-gel detection of abzyme activity (zymogram) [Bilyy R, Tomin A, et al. Antibody- mediated sialidase activity in blood serum of patients with multiple myeloma. J Mol Recognit 201 1 ; 24:576-84]. IgG fractions containing abzymes with sialidase activity were treated with pepsin to obtain F(ab)2 fragments which would maintain the sialidase activity but due to the lack of the Fc region would not be able to opsonize the treated cells.

Example 4

Enzyme Kinetics

[0116] The enzyme kinetics of the catalytically-active antibodies with sialidase activity obtained from immunized rabbits by the methods described herein was analyzed. As shown in Figure 4. (A) are the results of determining sialidase activity in serum samples of rabbits in the control group (1), immunoglobulin fractions of rabbits immunized with a hapten (2), and class-IgG immunoglobulin fractions of rabbits immunized with a hapten (3).

[0117] For the preparations obtained of catalytically-active IgG antibodies with sialidase activity, the kinetic parameters of the sialidase reaction were studied using 4- methylumbelliferyl-a-D-N-acetylneuraminic acid (4-MU-NANA) as a substrate, as previously described [Bilyy et al., Antibody-mediated sialidase activity in blood serum of patients with multiple myeloma. J Mol Recognit, 201 1 24:576-84]. The analysis results for the kinetic properties of the sialidase reaction catalyzed with purified class-IgG immunoglobulins are presented in Figures 4(B) and 4(C). The parameters determined for the kinetic reaction are: Km = 67.7 μΜ and Vmax = 0.02818 μΜ/min/mg. Example 5

Uses of the catalytic antibodies

[0118] The desialyation activity of the catalytically-active antibodies obtained from immunized rabbits by the methods described herein was analyzed. Human lymphocytes of the Jurkat line were cultured in a RPMI-1640 medium (Flow Laboratories, Scotland) in the presence of de-complemented fetal calf blood serum (Sigma, USA) and 50 μg/ml of gentamicin (Sigma, USA). Every two to three days, the cells passed through dilutions of the cell suspension in a ratio of 1 :5-l :2. The cells were cultivated in a humid atmosphere at 37°C and 5% C0₂. If necessary, apoptosis was induced in cells irradiated with ultraviolet type-B light, 180 mJ/cm2, with a subsequent period of incubation for 14 hours in a complete culture medium. The cells were rinsed (to a final concentration of 3 million/ml) and mixed into Ringer's solution. The cells were processed with a preparation of catalytically-active antibodies with sialidase activity, 30 mU, for three hours at 37°C or with a preparation of neuraminidase from Clostridium perfringens, 30 mU, for three hours at 37°C. The cells were rinsed twice with Ringer's solution, and a solution of a-2,6-sialo-specific black-elder lectin (SNA) was added to the cells, marked with fluorescein isotiocyanate (FITC), to a final concentration of 5 g/ml and propidium iodide to a final concentration of 1 μg/ml. The cells were incubated for 30 min at 4°C and an analysis performed of the content of sialyl residues on surface cells with SNA lectin bonds. The population of necrotic cells was shown by positive staining with propidium iodide. The intensity of lectin bonding was determined for the parameter of mean fluorescence intensity of intact and apoptotic cells, whose normalized values are presented in Figure 5. The data obtained are evidence that both processing with neuraminidase (as a positive control) and processing with catalytically-active antibodies with sialidase activity (obtained by the methods described herein), lead to a decrease in the content of sialic acid residues on the surface of both intact and apoptotic cells.

Example 6

[0119] Catalytically active antibodies (abzymes) attract much attention due to few unique features: being clearly associated with some disorders (particularly autoimmune) where their role could be protective or pathogenic depending on the type of disease and abzymes characteristics. Recently we have discovered a novel class of catalytically active antibodies, possessing sialidase activity (sialidase abzymes, SA) [Bilyy R, et al, Antibody- mediated sialidase activity in blood serum of patients with multiple myeloma. J Mol Recognit 201 1 ; 24:576-84]. Abzymes bearing sialidase activity were found in patients with multiple myeloma [Bilyy R, et al., Antibody- mediated sialidase activity in blood serum of patients with multiple myeloma. J Mol Recognit 2011 ; 24:576-84] or SLE [Bilyy R, et al. Cell surface glycans at SLE: changes during cells death, utilization for disease detection and molecular mechanism underlying their modification. In: Mavragani C, ed. Autoimmune Disorder I Book 1 : INTECH, 2011 :89-1 10], but not in patients with rheumatoid arthritis, paraprotein syndrom or multiple sclerosis as well as in normal healthy donors (NHD).

[0120] It is generally accepted that impaired clearance of apoptotic cells and the accumulation of necrotic material in various tissues may cause chronic inflammatory autoimmune diseases like SLE [Munoz LE, et al. The role of defective clearance of apoptotic cells in systemic autoimmunity. Nat Rev Rheumatol 2010; 6:280-9]. Artificial desialylation of dying lymphocytes with bacterial neuraminidase greatly enhanced clearance by human monocyte-derived macrophages (MoMa) of both apoptotic cells and apoptotic cells-derived microvesicles (ACMV) [Meesmann HM, et al. Decrease of sialic acid residues as an eat-me signal on the surface of apoptotic lymphocytes. J Cell Sci 2010; 123:3347]. Cellular sialidase Neul is activated during the execution phase of apoptosis and exposed on the surfaces of apoptotic cells/ ACMV as well as other intracellular molecules [Schiller M, et al. During apoptosis HMGB1 is translocated into apoptotic cell-derived membraneous vesicles. Autoimmunity; 2013 46(5):342-346]. At the cell surface sialidase is responsible for the desialylated glycoprofile of dying cells [Bilyy RO, et al. Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles. J Biol Chem 2012; 287:496- 503] and their neighbors [Shkandina T, et al. Sweet kiss of dying cell: Sialidase activity on apoptotic cell is able to act toward its neighbors. Autoimmunity 2012; 45:574-578]. Recently it was demonstrated that sialylation of "eat-me" signals regulates their functions [Malagolini N, et al. Apoptotic cells selectively uptake minor glycoforms of vitronectin from serum. Apoptosis 2013 1 -12].

[0121] The inventors analyzed whether F(ab)2 fragments of sialidase-abzymes facilitate clearance of dying cells. Viable and aged human peripheral blood derived granulocytes served as prey for MoMa. The data demonstrated that F(ab)2 fragments of abzymes with sialidase activity significantly facilitate the clearance by human MoMa of apoptotic prey.

[0122] Treatment of viable and apoptotic (UV-B irradiated) Jurkat cells with abzymes with sialidase activity, their F(ab)2 fragments or Clostridium perfringens neuraminidase, all at 30 mU, 3 h incubation at 37°C in Ringer buffer revealed the decrease of 2,6-sialic acid residues (quantified by SNA-FITC lectin binding). MFI of lectin binding (normalized to untreated cells) was 68% and 78% for viable and for apoptotic cells treated with abzymes with sialidase activity, respectively. The relative MFI values for the treatments with F(ab)2 fragment of the abzyme and for C. perfringens neuraminidase were 84% / 87% (V/A) and 47% / 60% (V/A), respectively.

[0123] The sialyl-abzymes and their F(ab)₂ fragments were used to desialylate viable and apoptotic (aged 24h) human PMN cells and to estimate the effect of phagocytosis by human allogenic and autologous MoMa of PMN. As depicted in Figure 7, treatment of PMN with F(ab)₂ fragments of abzymes with sialidase activity isolated from blood serum of patients with SLE significantly increased the prey count bound and ingested by autologous MoMa. Similarly, treatment of PMN with F(ab)₂ fragments of abzymes with sialidase activity isolated form immunized rabbits also significantly increased the clearance of apoptotic prey.

[0124] The ability of catalytically active antibodies, endowed with sialidase activity, to facilitate the clearance of apoptotic cells by human macrophages opens possibilities for the development of new therapeutic strategies for clearance-associated disorder.

[0125] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features. [0126] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0127] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[0128] In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0129] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0130] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

[0131] It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Claims

WHAT IS CLAIMED IS:

1. A method for producing an abzyme having sialidase activity comprising providing an inhibitor of neuraminidase as an antigen and immunizing an animal with the antigen so as to produce antibodies having sialidase activity.

2. The method of claim 1, wherein the inhibitor further comprises a bridging group and is any one or more of a carboxyl group, an amino group, an aldehyde group, a sulfhydryl group or a combination thereof.

3. The method of claim 2, wherein the inhibitor further comprises a carrier wherein the carrier is attached to the inhibitor via the bridging group.

4. The method of claim 3, wherein the carrier is any one or more of polyvinylpyrrolidone (PVP), bovine gamma-globulin (BGG), isologous mouse immunoglobulin (MIG), albumin, prealbumin, ox serum albumin, Megathura crenulata hemocyanin,

' Concholepas concholepas hemocyanin (CCH), Blue Carrier Protein, polyethylene glycol, polyacrylamide, agarose, purified protein derivative (PPD), KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), Cationized BSA, OVA (Ovalbumin), gelatin, multiple antigen eptides (MAPs), synthetic matrices or combinations thereof.

5. The method of claim 1, wherein the inhibitor of neuraminidase is an inhibitor of Neul, Neu2, Neu3 or Neu4.

6. The method of claim 1, wherein the inhibitor of neuraminidase binds to the active center of neuraminidase.

7. The method of claim 1, wherein the inhibitor of neuraminidase is a small molecule.

8. The method of claim 7, wherein the inhibitor of neuraminidase is a hapten.

9. The method of claim 8, wherein the neuraminidase inhibitor is any one or more of zanamivir, oseltamivir, siastatin B, laninamivir, peramivir, 2,3 -deny dro-2-deoxy-N- acetylneuraminic acid (DANA) or a variant thereof.

10. An abzyme or a fragment thereof or a derivative thereof having sialidase activity.

1 1. An abzyme produced by the method of claim 1 or a fragment, variant or derivative thereof.

12. The abzyme of claim 10 or 11, wherein the abzyme is selected from the group consisting of monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, chimeric antibodies, humanized antibodies, human antibodies and a single chain antibody.

13. The abzyme or fragment thereof of claim 10 or 1 1, wherein the fragment is produced by treating the abzyme with a protease.

14. The abzyme of claim 13, wherein the fragment is any one or more of scFv, Fc, F(ab) or F(ab)₂.

15. A method for treating autoimmune disease in a subject in need thereof comprising:

(i) providing a composition comprising an abzyme having sialidase activity; and

(ii) administering an effective amount of the composition to the subject so as to treat autoimmune disease in the subject.

16. A method for increasing clearance of apoptotic cell in a subject in need thereof comprising:

(i) providing a composition comprising an abzyme having sialidase activity; and

(ii) administering an effective amount of the composition to the subject so as to increase clearance of apoptotic cells in the subject.

17. A method for treating a disease-state resulting from reduced clearance of apoptotic cells in a subject in need thereof comprising:

(i) providing a composition comprising an abzyme having sialidase activity; and (ii) administering an effective amount of the composition to the subject so as to treat a disease-state resulting from reduced clearance of apoptotic cells in the subject.

18. The method of claim 15, 16 or 17, wherein the abzyme is selected from the group consisting of monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, chimeric antibodies, humanized antibodies, human antibodies and a single chain antibody.

19. The method of claim 15, 16 or 17, wherein the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof.

20. The method of claim 19, wherein the targeting element is fused to the abzyme or a fragment thereof.

21. The method of claim 19, wherein the targeting element is conjugated to the abzyme or a fragment thereof.

22. The method of claim 20 or 21, wherein the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof.

23. The method of claim 20 or 21, wherein the targeting element targets any one or more of negatively charged lipids, phosphatidylserine (PS), alpha-D-mannose-specific plasma membrane glycoepitopes, beta-D-galactose-specific plasma membrane glycoepitope, Annexin I, calreticulin, nucleolin, P ARP- 1 , APO- 1 , or a combination thereof.

24. A pharmaceutical composition comprising an abzyme having sialidase activity and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 18, wherein the abzyme is selected from the group consisting of monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, chimeric antibodies, humanized antibodies, human antibodies and a single chain antibody.

26. The pharmaceutical composition of claim 24, further comprising a targeting element to target apoptotic cells, cancer cells or a combination thereof.

27. The pharmaceutical composition of claim 26, wherein the targeting element is fused to the abzyme or a fragment thereof.

28. The pharmaceutical composition of claim 26, wherein the targeting element is conjugated to the abzyme or a fragment thereof.

29. The pharmaceutical composition of claim 27 or 28, wherein the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof.

30. The pharmaceutical composition of claims 27 or 28, wherein the targeting element targets any one or more of negatively charged lipids, phosphatidylserine (PS), alpha-D- mannose-specific plasma membrane glycoepitopes, beta-D-galactose-specific plasma membrane glycoepitopes, Annexin I, calreticulin, nucleolin, PARP-1, APO-1, or a combination thereof.

31. A kit comprising

(i) a composition comprising an abzyme having sialidase activity; and

(ii) instructions for use of the composition for the treatment of autoimmune disorders, treatment of disease-states resulting from reduced clearance of apoptotic cells or increasing clearance of apoptotic cells in a subject.

32. The kit of claim 31, wherein the abzyme is selected from the group consisting of monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, chimeric antibodies, humanized antibodies, human antibodies and a single chain antibody.

33. The kit of claim 31, wherein the composition further comprises a targeting element to target apoptotic cells, cancer cells or a combination thereof.

34. The kit of claim 33, wherein the targeting element is fused to the abzyme or a fragment thereof.

35. The kit of claim 33, wherein the targeting element is conjugated to the abzyme or a fragment thereof.

36. The kit of claim 34 or 35, wherein the targeting element is any one or more of peptides, proteins, antibodies, small molecules, oligonucleotides, nucleic acids or a combination thereof.

37. The kit of claim 34 or 35, wherein the targeting element targets any one or more of negatively charged lipids, phosphatidylserine (PS), alpha-D-mannose-specific plasma membrane epitopes, beta-D-galactose-specific plasma membrane glycoepitopes, Annexin I, calreticulin, nucleolin, PARP-1, APO-1, or a combination thereof.