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Definitions

• Acute or chronic inflammation is a common component of many clinical disorders and the complement system has been associated with a growing number of inflammatory conditions that include degenerative diseases, cancer and transplant rejection.
• the complement system acts as a sensor of pathogens, recognizes diseased and damaged host cells, and closely collaborates with other immune and defense systems to eliminate potential danger.
• insufficient, excessive, or poorly controlled complement activation can tip the balance between health and disease and lead to self-attack on host cells.
• Such an immune imbalance may fuel a cycle between complement, inflammatory cells, and tissue damage that recreates inflammatory stimulators rather than resolving them and exacerbates clinical complications. Inappropriate activation of complement has been linked to many
• the present disclosure is generally directed to anti-Clq Fab fragments and uses thereof.
• Complement is a central component of the innate immune system, especially as it relates to inflammation and the body's defense against invading organisms. Complement is also involved in the clearance of self-antigens and apoptotic cells, forms a bridge to adaptive immunity, and also plays a significant role in tissue regeneration and tumor growth.
• the complement system relies on an interplay of soluble and cell- surface-bound proteins that interact with pathogen cell surfaces to mark them for destruction by phagocytes.
• the complement system is made up of a large number of distinct plasma proteins, primarily produced by the liver. A number of these proteins are a class of proteases known as zymogens, which are themselves activated by proteolytic cleavage. These zymogens may be widely distributed without being active until detecting a local pathogen. The complement system thus is activated through a triggered enzyme cascade.
• Complement activation is initiated through three pathways: classical, alternative and lectin pathways. All three pathways are based on the detection of surface structures by pattern recognition proteins. In addition, all three pathways merge through a common intersection, complement C3. C3 is an acute phase reactant. The liver is the main site of synthesis, although small amounts are also produced by activated monocytes and
• a single chain precursor (pro-C3) of approximately 200 kD is found intracellularly; the cDNA shows that it comprises 1,663 amino acids. This is processed by proteolytic cleavage into alpha and beta subunits which in the mature protein are linked by disulfide bonds.
• Pro-C3 contains a signal peptide of 22 amino acid residues, the beta chain (645 residues) and the alpha chain (992 residues). The 2 chains are joined by 4 arginine residues that are not present in the mature protein.
• the classical pathway is activated by the binding of the complement protein Clq directly with patches of surface-bound antibodies (IgM and IgG), and also binding to C- reactive protein, serum amyloid P, pentraxin 3, and other ligands on the surface of apoptotic or microbial cells.
• Clq is a large multimeric protein of 460 kDa consisting of 18 polypeptide chains (6 Clq A chains, 6 Clq B chains, and 6 Clq C chains).
• Clr and Cls complement proteins bind to the Clq tail region to form the CI complex. Binding of the Clq complex to the surface of a cell or to the complement binding domain of an antibody Fc region induces a conformational change in Clq that leads to activation of an autocatalytic enzymatic activity in Clr, which then cleaves Cls to generate an active serine protease. Once activated, Cls cleaves C4, etc., leading to the complement cascade sequence. Ultimately this pathway leads to the formation of a membrane attack complex which lyses and kills the affected cell.
• Complement is nonspecific in that it can attack both foreign invaders and host cells.
• host cells including neurons
• Cl-INH dissociates Clr and Cls from the active CI complex, which protects host cells from lysis or damage from the membrane attack complex.
• Other proteins that protect from potential complement-mediated damage include C4b-binding protein (C4BP), factor H (FH), complement receptor 1 (CR1; CD35), complement receptor Ig (CRIg), decay accelerating factor (DAF; CD55), membrane cofactor protein (MCP; CD46), and CD59.
• C4BP C4b-binding protein
• FH factor H
• CR1 complement receptor 1
• CIg complement receptor Ig
• DAF decay accelerating factor
• MCP membrane cofactor protein
• CD59 membrane cofactor protein
• complement components are expressed by neurons and glial cells in vitro and in vivo. While their function in the brain is unknown, the expression of many of these complement proteins is upregulated by serum or inflammatory cytokines after brain injury or during the course of neurodegenerative disease pathology. Astrocytes in culture have been reported to express Clq, Clr, Cls, C4, C2, and C3, as well as the more terminal proteins. Neurons have been reported to express C4 and C3. Clq was shown to be expressed in neuronal synapses and to mark these synapses for elimination. See, e.g., U.S. Patent Publication Nos. 2012/0195880 and 2012/328601.
• complement factors such as Clq
• Neurodegenerative diseases involving synapse loss and considered to be amenable to treatments aiming at the neutralization of complement factors such as Clq include Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Down syndrome, Parkinson's disease, Huntington's disease, and the like.
• anaphylatoxin C5a has been shown to contribute to tumor growth in mice. In these situations where complement has deleterious effects, it is desirable to modulate its activation by using appropriate complement inhibitors.
• complement inhibitors are protein drugs that are immunogenic and have poor tissue penetration. For example, the failure of the anti-C5 mAb pexelizumab (Alexion)
• Fab antibody fragments directed towards the inhibition of different components of the complement cascade may be advantageous for preventative, diagnostic, and therapeutic clinical applications.
• Such Fab molecules are desired in situations that require fast clearance, higher tissue penetration capability, absence of Fc-mediated effector functions (such as CDC, ADCC, and
• Fab molecules do not form cross- linked immunocomplexes that can trigger anaphylaxis.
• the present disclosure provides an antibody Fab fragment that binds to a protein in the complement cascade.
• the antibody Fab fragment binds to a Clq protein.
• the antibody Fab fragment comprises a heavy chain and a light chain, wherein the heavy chain is truncated after the first heavy chain domain.
• the antibody Fab fragment is an anti-Clq antibody Fab fragment.
• the antibody Fab fragment may be prepared by any suitable method known in the art.
• the antibody Fab fragment may be obtained from any whole antibody, especially a whole monoclonal antibody, using any suitable enzymatic cleavage and/or digestion techniques, such as treatment with the cysteine protease papain to yield an antibody Fab fragment.
• the antibody Fab fragment of the present invention is prepared by the use of recombinant DNA techniques involving the manipulation and re-expression of DNA encoding antibody variable and constant regions. Standard molecular biology techniques may be used to modify, add or delete further amino acids or domains as desired. Any alterations to the variable or constant regions are still encompassed by the terms 'variable' and 'constant' regions as used herein.
• PCR is used to introduce a stop codon immediately following the codon encoding the interchain cysteine of C H I, such that translation of the C H I domain stops at the interchain cysteine.
• Methods for designing suitable PCR primers are well known in the art and the sequences of antibody C H I domains are readily available.
• stop codons may be introduced using site-directed mutagenesis techniques.
• the antibody Fab fragment starting material of the present invention may be derived from any antibody isotype ("class") including for example IgG, IgM, IgA, IgD and IgE and subclasses thereof, including for example IgGl, IgG2, IgG3 and IgG4.
• class an antibody isotype
• the antibody Fab fragment of the present invention is derived from IgGl .
• the antibody Fab fragment starting material may be obtained from any species including for example mouse, rat, rabbit, pig, hamster, camel, llama, goat or human.
• the heavy and light chains of the antibody Fab fragment are from murine IgGl .
• Parts of the antibody Fab fragment may be obtained from more than one species.
• the antibody fragments may be chimeric or humanized.
• the constant regions are from one species and the variable regions from another.
• antibody Fab fragment is humanized.
• variable region of the antibody fragment has been created using recombinant DNA engineering techniques.
• engineered versions include those created for example from natural antibody variable regions by insertions, deletions or changes in or to the amino acid sequences of the natural antibodies.
• Particular examples of this type include those engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from one antibody and the remainder of the variable region domain from a second antibody.
• the antibody Fab fragment comprises a heavy chain of the amino acid sequence provided in SEQ ID NO: 1 and a light chain of the amino acid sequence provided in SEQ ID NO:2.
• the DNA sequence encoding the amino acids of SEQ ID NOs 1 and 2 are provided.
• the nucleic acid sequence encoding SEQ ID NO: 1 is SEQ ID NO:3.
• the nucleic acid sequence encoding SEQ ID NO:2 is SEQ ID NO:4.
• antibody Fab fragments wherein the heavy and light chains comprise a sequence having at least 90% identity or similarity to the sequences of SEQ ID NO: l and SEQ ID NO:2, respectively.
• the antibody Fab fragment comprises a heavy chain sequence having at least 90%, 95% or 98% identity or similarity to SEQ ID NO: 1 and a light chain sequence having at least 90%, 95% or 98% identity or similarity to SEQ ID NO:2.
• the antibody Fab fragment binds specifically to human Clq, mouse Clq, dog Clq, rhesus Clq, cynomolgus monkey Clq or rat Clq.
• the antibody Fab fragments binds specifically to both human Clq and mouse Clq. In some embodiments, the antibody Fab fragments binds specifically to both human Clq and rat Clq. In some embodiments, the antibody Fab fragments binds specifically to both human Clq and dog Clq. In some embodiments, the antibody Fab fragments binds specifically to both human Clq and rhesus Clq. In some embodiments, the antibody Fab fragments binds specifically to both human Clq and cynomolgus monkey Clq. In some embodiments, the antibody Fab fragments binds specifically to both mouse Clq and rat Clq. In some embodiments, the antibody Fab fragments binds specifically to both mouse Clq and dog Clq.
• the antibody Fab fragments binds specifically to both mouse Clq and rhesus Clq. In some embodiments, the antibody Fab fragments binds specifically to both mouse Clq and cynomolgus monkey Clq. In some embodiments, the antibody Fab fragments binds specifically to both rat Clq and dog Clq. In some embodiments, the antibody Fab fragments binds specifically to both rat Clq and rhesus Clq. In some embodiments, the antibody Fab fragments binds specifically to both rat Clq and cynomolgus monkey Clq. In some embodiments, the antibody Fab fragments binds specifically to both dog Clq and rhesus Clq.
• the antibody Fab fragments binds specifically to both dog Clq and cynomolgus monkey Clq. In some embodiments, the antibody Fab fragments binds specifically to rhesus dog Clq and cynomolgus monkey Clq. In some embodiments, the antibody Fab fragment binds to human Clq, mouse Clq and/or rat Clq, and at least one of the following: dog Clq, rhesus Clq, and cynomolgus monkey Clq. In some embodiments, the antibody Fab fragment binds to both human Clq and mouse Clq. In other embodiments, the antibody Fab fragment binds to human Clq, mouse Clq, rat Clq, dog Clq, rhesus Clq, and cynomolgus monkey Clq.
• the antibody Fab fragment binds essentially the same Clq epitope as the antibody Ml produced by the hybridoma cell line with ATCC Accession Number PTA-120399. In some cases, the antibody Fab fragment inhibits the binding of the monoclonal antibody Ml produced by a hybridoma cell line with ATCC Accession Number PTA-120399 to human Clq or to mouse Clq.
• a humanized anti-Clq antibody Fab fragment that binds to an epitope of Clq that is the same as or overlaps with the Clq epitope bound by another antibody of this disclosure.
• the humanized anti- Clq antibody Fab fragment that binds to an epitope of Clq is the same as or overlaps with the Clq epitope bound by anti-Clq antibody Ml produced by the hybridoma cell line with ATCC Accession Number PTA-120399.
• the humanized anti-Clq antibody Fab fragment competes with another antibody of this disclosure for binding to Clq.
• the anti-Clq antibody Fab fragment competes with anti-Clq antibody Ml produced by the hybridoma cell line with ATCC Accession Number PTA- 120399 or anti-Clq binding fragments thereof.
• the antibody Fab fragments of the present disclosure may comprise the heavy and light chain CDR3s of variable regions of the monoclonal antibody Ml produced by the hybridoma cell line having ATCC Accession Number PTA-120399.
• the antibody Fab fragment further comprises the CDR2s of variable regions of the monoclonal antibody Ml .
• the antibody Fab fragment further comprises the CDRs of variable regions of the monoclonal antibody Ml .
• the antibody Fab fragment may further comprise any combinations of the CDRs.
• the antibody Fab fragment has been engineered to increase brain penetration. In some cases, the antibody Fab fragment has better brain penetration as compared to the corresponding full-length antibody.
• the antibody Fab fragment has only one binding site. In some embodiments, the antibody Fab fragment has the same affinity for Clq as the whole Clq antibody, incorporated herein by reference from US Pat No: 62/075793. In some embodiments, the antibody Fab fragment inhibits proteins of the complement cascade, e.g., Clq, C4, C2, C3 convertase, C3a, C5, C3b, C5b, C6, C7, C8, and/or C9.
• the antibody fragment has a shorter half-life in human circulation as compared to its corresponding full-length antibody.
• the antibody Fab fragment is injected directly into the eye. In some embodiments, the antibody Fab fragment is injected into the eye for prevention or treatment of an ocular disease or condition and may be typically administered by ocular, intraocular, and/or intravitreal injection. Other methods administration by also be used, which includes but is not limited to, topical, parenteral, subcutaneous, intraperitoneal,
• Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.
• the antibody Fab fragments of the present invention are useful for the prevention and treatment complement-associated eye conditions (all eye conditions and diseases the pathology of which involves complement, including the classical and the alternative pathways), such as, for example, macular degenerative diseases, such as Chronic open-angle glaucoma, acute closed angle glaucoma, all stages of age-related macular degeneration (AMD), including dry and wet (non-exudative and exudative) forms (AMD-wet), Geographic atrophy, choroidal neovascularization (CNV), uveitis, diabetic and other ischemia-related retinopathies, endophthalmitis, and other intraocular neovascular diseases, such as diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization, retinal
• macular degenerative diseases such as Chronic open-angle glaucoma, acute
• neovascularization Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, ANCA vasculitis, Purtscher retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, and multiple sclerosis.
• a preferred group of complement-associated eye conditions includes age-related macular degeneration (AMD), including non-exudative (wet) and exudative (dry or atrophic) AMD, choroidal neovascularization (CNV), diabetic retinopathy (DR), and endophthalmitis.
• AMD age-related macular degeneration
• CNV choroidal neovascularization
• DR diabetic retinopathy
• the antibody Fab fragment has a dissociation constant (K D ) for human Clq that ranges from 10 pM to 20 pM, or 1 pM to less than 10 pM. In some embodiments, the antibody Fab fragment has a dissociation constant (K D ) for mouse Clq that ranges from 1 pM to 200 pM. In some embodiments, the antibody Fab fragment specifically binds to and inhibits a biological activity of Clq.
• the biological activity is (1) Clq binding to an autoantibody, (2) Clq binding to Clr, (3) Clq binding to Cls, (4) Clq binding to phosphatidylserine, (5) Clq binding to pentraxin-3, (6) Clq binding to C-reactive protein (CRP), (7) Clq binding to globular Clq receptor (gClqR), (8) Clq binding to complement receptor 1 (CR1), (9) Clq binding to beta-amyloid, (10) Clq binding to calreticulin (11) Clq binding to apoptotic cells, or (12) Clq binding to components of a nerve cell membrane.
• the biological activity is (1) activation of the classical complement activation pathway, (2) activation of antibody and complement dependent cytotoxicity, (3) CH50 hemolysis, (4) synapse loss, (5) B-cell antibody production, (6) dendritic cell maturation, (7) T-cell proliferation, (8) cytokine production (9) microglia activation, (10) Arthus reaction, (11) phagocytosis of synapses or nerve endings, or (12) activation of complement receptor 3 (CR3/C3) expressing cells.
• CR3/C3 complement receptor 3
• CH50 hemolysis comprises human, mouse, and/or rat CH50 hemolysis.
• the antibody Fab fragment is capable of neutralizing from at least about 50%, to at least about 95% of CH50 hemolysis. In some embodiments, the antibody Fab fragment is capable of neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng, less than 100 ng, less than 50 ng, or less than 20 ng. In some embodiments, the antibody Fab fragment binds Clq and inhibits biological function with a binding stoichiometry that ranges from less than 6: 1 to 1 : 1, or less than 2: 1 to 1 : 1.
• the antibody Fab fragment is humanized.
• the present disclosure provides for an isolated host cell comprising a nucleic acid sequence of any of the preceding embodiments.
• the host comprises a cloning or expression vector comprising the nucleic acid sequences of the antibody Fab fragment.
• the host cell is cultured containing the expression vector and nucleic acid in conditions suitable for expression of the antibody Fab fragment.
• the antibody Fab fragment is subsequently recovered from the host cell (or host cell medium).
• compositions are provided containing anti-Clq antibody Fab fragments in combination with pharmaceutically acceptable carriers.
• the present disclosure also provides a kit containing an anti-Clq antibody Fab fragment for use in any of the methods described herein.
• the present disclosure provides for a method of treating or preventing a disease associated with complement activation in an individual in need of such treatment, the method comprising administering an anti-Clq antibody Fab fragment.
• the disease associated with complement activation is a neurodegenerative disorder.
• the neurodegenerative disorder is associated with the loss of synapses or nerve connections, such as synapse loss that is dependent on the complement receptor 3(CR3)/C3 or complement receptor CR1.
• the neurodegenerative disorder is associated with pathological activity-dependent synaptic pruning, synapse phagocytosis by microglia.
• the neurodegenerative disorder is associated with Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barre ' syndrome (GBS), Myasthenia Gravis, Bullous Pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, and Huntington's disease.
• the disease associated with complement activation is an inflammatory disease, an autoimmune disease, or metabolic disorder selected from diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resultant
• glomerulonephritis and vasculitis cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulemia, antiphospholipid syndrome, Chronic open-angle glaucoma, acute closed angle glaucoma, macular degenerative diseases, age-related macular degeneration (AMD), (AMD-wet), Geographic atrophy choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Neuromyelitis Optica (NMO), Central Retinal Vein Occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's heredit
• the disease associated with complement activation is an autoimmune disease selected from myasthenia gravis, Diabetes mellitus type 1, Hashimoto's thyroiditis, Addison's disease, Coeliac disease, Crohn's disease, pernicious anaemia, Pemphigus vulgaris, vitiligo, autoimmune hemolytic anemias, paraneoplastic syndromes, a vasculitis disease, hypocomplementemic urticarial vasculitis (HUV), polymyalgia rheumatica, temporal arteritis, Wegener's granulomatosis, multiple sclerosis, Guillain-Barre syndrome, Myasthenia Gravis, Bullous Pemphigoid, or myositis.
• autoimmune disease selected from myasthenia gravis, Diabetes mellitus type 1, Hashimoto's thyroiditis, Addison's disease, Coeliac disease, Crohn's disease, pernicious anaemia, Pemphigus vulgaris, viti
• the present disclosure provides a method of detecting synapses in an individual, by a) administering an anti-Clq antibody Fab fragment to the individual, and b) detecting antibody Fab fragment bound to synapses, thereby detecting synapses in the individual.
• the antibody Fab fragment bound to synapses is detected using imaging techniques selected from positron emission tomography (PET), X-ray computed tomography, single-photon emission computed tomography
• the detection of antibody Fab fragment bound to synapses provides a quantitative measure of the number of synapses in the individual, such that the number of synapses in the individual is measured repeatedly over a period of time and a loss of synapses in the individual is detected over time and the loss of synapses over time is a measure for the efficacy of a treatment for the neurodegenerative disease or autoimmune disease.
• the present disclosure provides a method of detecting synapses in a biological sample, by a) contacting the biological sample with a humanized anti-Clq antibody Fab fragment, and b) detecting antibody Fab fragment bound to synapses, thereby detecting synapses in the individual.
• the method further comprises a step before step a) of obtaining the biological sample from an individual.
• the biological sample comprises a biopsy specimen, a tissue, or a cell.
• the antibody Fab fragment is detected by immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micro-positron emission tomography.
• Identity indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.
• similarity indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
• leucine may be substituted for isoleucine or valine.
• Other amino acids which can often be substituted for one another include but are not limited to:
• the antibody Fab fragments may be useful in the detection or treatment of many diseases or disorders, such as neurodegenerative disorders, such as neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barre ' syndrome (GBS),
• GNS Guillain-Barre ' syndrome
• the antibody Fab fragments may be useful in the detection or treatment of inflammatory disease, autoimmune disease, or metabolic disorder, such as diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resultant glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulemia, antiphospholipid syndrome, Chronic open-angle glaucoma, acute closed angle glaucoma, macular degenerative diseases, age-related macular degeneration (AMD), (AMD-wet), Geographic atrophy choroidal neovascularization (CNV),
• inflammatory disease such as diabetes, obesity, rheuma
• methods are provided for protecting or treating an individual suffering from adverse effects of synapse loss.
• neurodegenerative conditions involving synaptic loss which conditions may include Alzheimer's disease; amyotrophic lateral sclerosis; multiple sclerosis, glaucoma, myotonic dystrophy, Down syndrome; Parkinson's disease, Huntington's disease; and the like.
• the loss of synapses is inhibited by contacting neurons with agents that block complement, including specific components, such as Clq.
• a method is provided of determining a subject's risk of developing a disease associated with complement activation, comprising: (a) administering an anti-Clq antibody fragment to the subject, wherein the antibody fragment is coupled to a detectable label; (b)detecting the detectable label to measure the amount or location of Clq in the subject; and (c) comparing the amount or location of Clq to a reference, wherein the risk of developing a disease associated with complement activation is characterized based on the comparison of the amount of Clq as compared to the reference.
• the detectable label comprises a nucleic acid
• the antibody is labeled with a coenzyme such as biotin using the process of biotinylation.
• biotin is used as a label
• the detection of the antibody is accomplished by addition of a protein such as avidin or its bacterial counterpart streptavidin, either of which can be bound to a detectable marker such as the aforementioned dye, a fluorescent marker such as fluorescein, a radioactive isotope or an enzyme such as peroxidase.
• the detectable label is detected using an imaging agent for x- ray, CT, MRI, ultrasound, PET and SPECT.
• the fluorescent label is selected from fluorescein, rhodamine, cyanine dyes or BODIPY.
• a method is provided of reducing a subject's risk of developing a disease associated with complement activation, comprising administering an anti-Clq antibody fragment, wherein the anti-Clq antibody fragment prevents or reduces the risk of developing a disease associated with complement activation, thereby preventing or reducing the risk of future diseases associated with complement activation.
• the antibody Fab fragment has a shorter half-life as compared to its corresponding full-length antibody.
• Figure 1 consists of two panels, (A) and (B).
• Figure 1 shows that the Ml Fab has the same binding affinity as the Ml whole antibody.
• Figure 1A shows an ELISA measuring antibody binding to Clq (anti -kappa light chain detection).
• Figure IB is an image showing Ml whole antibody (bivalent) and Ml Fab (monovalent).
• Figure 2 shows that the Ml Fab has the same functional potency as the Ml whole antibody.
• Figure 2 shows a standard functional measure of Clq activity inhibition of RBC lysis.
• Ig immunoglobulin
• proteolytic enzymes that cleave polypeptide sequences have been used to dissect the structure of antibody molecules and to determine which parts of the molecule are responsible for its various functions.
• Limited digestion with the protease papain cleaves antibody molecules into three fragments. Two fragments, known as Fab fragments, are identical and contain the antigen- binding activity.
• the Fab fragments correspond to the two identical arms of the antibody molecule, each of which consists of a complete light chain paired with the V H and C H 1 domains of a heavy chain. The other fragment contains no antigen binding activity but was originally observed to crystallize readily, and for this reason was named the Fc fragment (Fragment crystallizable).
• Fab molecules were compared to IgG molecules, it was found that Fab are superior to IgG for certain in vivo applications due to their higher mobility and tissue penetration capability, their reduced circulatory half-life, their ability to bind antigen monovalently without mediating antibody effector functions, and their lower immunogenicity.
• the Fab molecule is an artificial ⁇ 50-kDa fragment of the Ig molecule with a heavy chain shortened by constant domains C H 2 and C H 3.
• Two heterophilic (V L -V H and C L -C H 1) domain interactions underlie the two-chain structure of the Fab molecule, which is further stabilized by a disulfide bridge between C L and C H I .
• Fab and IgG have identical antigen binding sites formed by six complementarity-determining regions (CDRs), three each from V L and V H (LCDRl, LCDR2, LCDR3 and HCDRl, HCDR2, HCDR3).
• the CDRs define the hypervariable antigen binding site of antibodies.
• LCDR3 and HCDR3 typically form the core of the antigen binding site.
• the conserved regions that connect and display the six CDRs are referred to as framework regions.
• the framework regions form a sandwich of two opposing antiparallel ⁇ -sheets that are linked by hypervariable CDR loops on the outside and by a conserved disulfide bridge on the inside.
• the present disclosure provides an anti-Clq antibody Fab fragment that binds to a Clq protein comprising a heavy (V H /C H 1) and light chain (V L /C L ), wherein the anti-Clq antibody Fab fragment has six complementarity determining regions
• CDRs three each from V L and V H (HCDRl, HCDR2, HCDR3, and LCDRl, LCDR2,
• the heavy chain of the antibody Fab fragment is truncated after the first heavy chain domain of IgGl (SEQ ID NO: l), and comprises the following amino acid sequence:
• CDRs complementarity determining regions
• CDRs complementarity determining regions
• the light chain domain of the antibody Fab fragment comprises the following amino acid sequence (SEQ ID NO:2):
• CDRs complementarity determining regions
• SEQ ID NO:4 The nucleotide sequence corresponding to SEQ ID NO:2 is SEQ ID NO:4:
• CDRs complementarity determining regions
• amino acid sequence corresponding to SEQ ID NO:5 is:
• amino acid sequence corresponding to SEQ ID NO:6 is:
• SEQ ID NO: 6 OVOLVOSGAELKKPGASVKVSCKSSGYHFTSYWMHWVKQAPGQGLEWIGVIHPN SGSINYNEKFESRATITVDKSTSTAYMELSSLRSEDTAVYYCAGERDSTEVLPMDY WGQGTTVTVSS (SEQ ID NO: 6).
• the complementarity determining regions (CDRs) of SEQ ID NO: 6 are depicted in bolded and underlined text.
• amino acid sequence corresponding to SEQ ID NO: 7 is:
• SEQ ID NO: 7 OVOLVOSGAELKKPGASVKVSCKSSGYHFTSY MHWVKQAPGOGLEWIGVIHPN SGSINYNEKFESRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGERDSTEVLPMDY WGQGTTVTVSS (SEQ ID NO: 7).
• the complementarity determining regions (CDRs) of SEQ ID NO: 7 are depicted in bolded and underlined text.
• amino acid sequence corresponding to SEQ ID NO:8 is:
• SEQ ID NO: 8 OVOLVOSGAELKKPGASVKVSCKSSGYHFTSYWMHWVRQAPGOGLEWIGVIHPN SGSINYNEKFESRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGERDSTEVLPMDY WGQGTTVTVSS (SEQ ID NO: 8).
• the complementarity determining regions (CDRs) of SEQ ID NO:8 are depicted in bolded and underlined text.
• amino acid sequence corresponding to SEQ ID NO:9 is:
• DVOITOSPSYLAASLGERATINCRASKSINKYLAWYQOKPGKTNKLLIYSGSTLOSG IPARFSGSGSGTDFTLTISSLEPEDFAMYYCOOHNEYPLTFGOGTKLEIK
• CDRs complementarity determining regions
• amino acid sequence corresponding to SEQ ID NO: 10 is:
• CDRs complementarity determining regions
• amino acid sequence corresponding to SEQ ID NO: 1 1 is:
• SEQ ID NO: 11 The complementarity determining regions (CDRs) of SEQ ID NO: 11 are depicted in bolded and underlined text.
• the amino acid sequence corresponding to SEQ ID NO: 12 is:
• SEQ ID NO: 12 DIQLTQ SP S SL S ASLGERATINCRASKSINKYLA W YQQKPGKAPKLLI YSGSTLQSGI P ARF S GS GS GTDF TLTIS SLEPEDF AM Y YC QQHNE YPLTF GQGTKLEIK (SEQ ID NO: 12).
• CDRs complementarity determining regions
• Methods are provided for protecting or treating an individual suffering from adverse effects of synapse loss. It is shown herein that immature astrocytes in normal development produce a signal that induces neurons to express specific complement proteins, thus enabling a developmental window during which synapse elimination occurs. Expression of these proteins in development mirrors the period of developmental synaptogenesis, being off in embryonic brain and adult brain but on at high levels in postnatal brain.
• synapse loss is inhibited by contacting neurons with inhibitors or antagonists of the complement pathway.
• inhibitors can block activation of the complement cascade, can block the expression of specific complement proteins in neurons, can interfere with signaling molecules that induce complement activation, can upregulate expression of complement inhibitors in neurons, and otherwise interfere with the role of complement in synapse loss.
• the ability to prevent synapse loss, e.g. in adult brains, has important implications for maintaining normal neuronal function in a variety of neurodegenerative conditions.
• a or “an” may mean one or more.
• the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
• reference to an "antibody” is a reference to from one to many antibodies, and includes equivalents thereof known to those skilled in the art, and so forth.
• another may mean at least a second or more.
• preventing is art-recognized, and when used in relation to a condition, such as an epilepsy disease, is well understood in the art, and includes administration of a composition which reduces the frequency or severity, or delays the onset, of one or more symptoms of the medical condition in a subject relative to a subject who does not receive the composition.
• the prevention of epilepsy disease progression includes, for example, slowing or halting the average amount of neurodegeneration in a population of patients receiving a therapy relative to a control population that did not receive the therapy, e.g., by a statistically and/or clinically significant amount.
• the prevention of neurodegenerative disease progression includes reducing the likelihood that a patient receiving a therapy will develop a disability, such as cognitive decline and/or memory loss, or delaying the onset of disability, relative to a patient who does not receive the therapy.
• subject refers to a living mammal and may be
• mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
• the term does not denote a particular age or gender.
• treating includes reducing, arresting, or reversing the symptoms, clinical signs, or underlying pathology of a condition to stabilize or improve a subject's condition or to reduce the likelihood that the subject's condition will worsen as much as if the subject did not receive the treatment.
• terapéuticaally effective amount of a compound with respect to the subject method of treatment refers to an amount of the compound(s) in a preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit/risk ratio applicable to any medical treatment.
• a therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual.
• an individual “at risk” of developing a particular disease, disorder, or condition may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein.
• At risk denotes that an individual has one or more risk factors, which are measurable parameters that correlate with development of a particular disease, disorder, or condition, as known in the art. An individual having one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition than an individual without one or more of these risk factors.
• Chronic administration refers to administration of the medicament(s) in a continuous as opposed to acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
• Intermittent refers to treatment that is not administered consecutively without interruption, but rather is cyclic/periodic in nature.
• administration injointly with another compound or composition includes simultaneous administration and/or administration at different times. Conjoint administration also encompasses administration as a co-formulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.
• Synapse loss are asymmetric communication junctions formed between two neurons, or, at the neuromuscular junction (NMJ) between a neuron and a muscle cell.
• Chemical synapses enable cell-to-cell communication via secretion of neurotransmitters, whereas in electrical synapses signals are transmitted through gap junctions, specialized intercellular channels that permit ionic current flow.
• other molecules that modulate synaptic function can diffuse through gap junctional pores.
• pre- and postsynaptic membranes are separated by a synaptic cleft containing extracellular proteins that form the basal lamina. Synaptic vesicles are clustered at the presynaptic release site, transmitter receptors are clustered in junctional folds at the postsynaptic membrane, and glial processes surround the nerve terminal.
• Synaptogenesis is a dynamic process. During development, more synapses are established than ultimately will be retained. Therefore, the elimination of excess synaptic inputs is a critical step in synaptic circuit maturation. Synapse elimination is a competitive process that involves interactions between pre- and postsynaptic partners. In the CNS, as with the NMJ, a developmental, activity-dependent remodeling of synaptic circuits takes place by a process that may involve the selective stabilization of coactive inputs and the elimination of inputs with uncorrected activity. The anatomical refinement of synaptic circuits occurs at the level of individual axons and dendrites by a dynamic process that involves rapid elimination of synapses. As axons branch and remodel, synapses form and dismantle with synapse elimination occurring rapidly.
• synapse loss is an early pathological event common to many neurodegenerative disorders, and is the best correlate to the cognitive impairment.
• AD Alzheimer's disease
• transgenic animal models have shown that synaptic damage occurs early in disease progression. This early disruption of synaptic connections in the brain results in neuronal dysfunction that, in turn, leads to the characteristic symptoms of dementia and/or motor impairment observed in several neurodegenerative disorders.
• ⁇ has a preferential localization at central and peripheral synaptic sites.
• abnormal expression of mutant forms of ⁇ results not only in amyloid deposition, but also in widespread synaptic damage. This synaptic pathology occurs early and is associated with levels of soluble ⁇ - 42 rather than with plaque formation.
• Other neurodegenerative diseases where gene products have been shown to be closely associated with synaptic complexes include Huntington's disease (HD) and myotonic dystrophy (DM). Huntingtin is a membrane-bound protein with a distribution very similar to that of synaptic vesicle protein synaptophysin.
• DMK serine/ threonine kinase
• Inhibiting synapse loss results in maintenance or reduced loss of synapses, where a decrease would otherwise occur.
• modulation of synapse loss as used herein, it is meant that the number of synapses lost is either enhanced or suppressed as required in the specific situation.
• modulator of synapse loss refers to an agent that is able to alter synapse loss. Modulators include, but are not limited to, both “activators” and “inhibitors”.
• An “activator” or “agonist” is a substance that enhances synapse loss.
• an “inhibitor” or “antagonist” decreases synapse loss. The reduction may be complete or partial.
• modulators include, without limitation, Clq antagonists and agonists.
• Agonists and antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules that decrease the effect of a protein.
• the term "analog” is used herein to refer to a molecule that structurally resembles a molecule of interest but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the starting molecule, an analog may exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher potency at a specific receptor type, or higher selectivity at a targeted receptor type and lower activity levels at other receptor types) is an approach that is well known in pharmaceutical chemistry.
• Complement is a system of plasma proteins that interacts with the cell surfaces of pathogens or cells to mark them for destruction by phagocytes.
• the complement system is made up of a large number of distinct plasma proteins, primarily produced by the liver. A number of these proteins are a class of proteases, called zymogens, which are themselves activated by proteolytic cleavage. These zymogens can thus be widely distributed without being active until activated by a local pathogen.
• the complement system thus is activated through a triggered enzyme cascade.
• Clq is a large multimeric protein of 460 kDa consisting of 18 polypeptide chains (6 Clq A chains, 6 Clq B chains, and 6 Clq C chains).
• Clr and Cls complement proteins to bind to the Clq tail region to form the CI complex. Binding of the Clq complex to the surface of a cell or to the complement binding domain of an antibody Fe region induces a conformational change in Clq that leads to activation of an autocatalytic enzymatic activity in Clr, which then cleaves Cls to generate an active serine protease.
• complement proteins are expressed by neurons and glial cells in vitro and in vivo. Their function in the brain is unknown. The expression of many of these complement proteins is upregulated by serum or inflammatory cytokines or after brain injury. Astrocytes in culture have been reported to express Clq, Clr, Cls, C4, C2, and C3, as well as the more terminal proteins. Neurons have been reported to express C4 and C3, but only to express Clq after brain injury.
• C3 is an acute phase reactant.
• the liver is the main site of synthesis, although small amounts are also produced by activated monocytes and macrophages.
• a single chain precursor (pro-C3) of approximately 200 kD is found intracellularly; the eDNA shows that it comprises 1,663 amino acids. This is processed by proteolytic cleavage into alpha and beta subunits which in the mature protein are linked by disulfide bonds.
• Pro-C3 contains a signal peptide of 22 amino acid residues, the beta chain (645 residues) and the alpha chain (992 residues).
• C3 undergoes spontaneous cleavage resulting in complement B binding to C3b.
• Diffusion of the Ba subunit results in an active alternative pathway C3 convertase (C3bBb).
• C3bBb is stabilized by binding to properdin prior to merging.
• complement Inhibition of complement.
• a number of molecules are known that inhibit the activity of complement.
• suitable inhibitors can be screened by methods described herein.
• normal cells can produce proteins that block complement activity, e.g., CD59, CI inhibitor, etc.
• complement is inhibited by upregulating expression of genes encoding such polypeptides.
• Modifications of molecules that block complement activation are also known in the art.
• Such molecules include, without limitation, modified complement receptors, such as soluble CRl .
• the mature protein of the most common allotype of CRl contains 1998 amino acid residues: an extracellular domain of 1930 residues, a transmembrane region of 25 residues, and a cytoplasmic domain of 43 residues.
• the entire extracellular domain is composed of 30 repeating units referred to as short consensus repeats (SCRs) or complement control protein repeats (CCPRs), each consisting of 60 to 70 amino acid residues.
• SCRs short consensus repeats
• CCPRs complement control protein repeats
• Clq binds specifically to human CRl .
• CRl recognizes all three complement opsonins, namely C3b, C4b, and Clq.
• sCRl recombinant human CRl
• cClqR human Clq receptors
• gClqR is not a membrane-bound molecule, but rather a secreted soluble protein with affinity for the globular regions of CI q, and may act as a fluid-phase regulator of complement activation.
• Decay accelerating factor (CD55) is composed of four SCRs plus a serine/threonine-enriched domain that is capable of extensive 0-linked glycosylation.
• OAF is attached to cell membranes by a glycosyl phosphatidyl inositol (GPI) anchor and, through its ability to bind C4b and C3b, it acts by dissociating the C3 and C5 convertases.
• GPI glycosyl phosphatidyl inositol
• Soluble versions of OAF (sDAF) have been shown to inhibit complement activation.
• CI inhibitor a member of the "serpin" family of serine protease inhibitors, is a heavily glycosylated plasma protein that prevents fluid-phase CI activation.
• CI inhibitor regulates the classical pathway of complement activation by blocking the active site of Clr and Cls and dissociating them from Clq.
• Peptide inhibitors of complement activation include C5a (van Oostrum et al., 1996); C5a C-terminal octapeptides (Kawai et al., 1992); C5a His67-modified C-terminal octapeptide analogues (Or et al., 1992); C089 (C5a hexapeptide, Konteatis et al., 1994 ); C3a C-terminus (Kretzschmar et al., 1992); Factor B-related hexapeptides (Lesavre et al., 1982); Clq B chain helical region (Fryer et al., 1997); DFP (Diisopropyl fluorophosphates, Cole et al., 1997); BCX-14 70 (K-76 analog, Kaufman et al., 1995); TKIX (K-76 derivative) Sindelar et al. 1996); K-76 derivative, Tanaka 1996); F
• inhibitory molecules include Fucan (Charreau et al., 1997); Complestatin (Momota et al., 1991 ); Decorin (Krumdieck et al., 1992); heparin (te Velthuis et al., 1996); LU 1198 (Gralinski et al., 1997); CSPG (Kirschfink et al., 1997); L-156,602 (Tsuji et al., 1992); CVFb (Jungi and McGregor, 1979); M5 (Chen and Rael, 1997).
• complement-associated eye condition is used in the broadest sense and includes all eye conditions the pathology of which involves complement, including the classical and the alternative pathways, and in particular the alternative pathway of
• Complement-associated eye conditions include, without limitation, Chronic open-angle glaucoma, acute closed angle glaucoma, macular degenerative diseases, such as all stages of age-related macular degeneration (AMD), including dry and wet (non-exudative and exudative) forms, choroidal neovascularization (CNV), uveitis, diabetic and other ischemia-related retinopathies, and other intraocular neovascular diseases, such as diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, ANCA vasculitis, Purtscher retinopathy, Sjogren's dry eye disease, dry AMD
• a preferred group of complement-associated eye conditions includes age-related macular degeneration (AMD), including non-exudative (wet) and exudative (dry or atrophic) AMD, choroidal neovascularization (CNV), diabetic retinopathy (DR), and endophthalmitis.
• AMD age-related macular degeneration
• CNV choroidal neovascularization
• DR diabetic retinopathy
• immunoglobulin (Ig) is used interchangeably with “antibody” herein.
• antibody herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies ⁇ e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
• the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. The pairing of a V H and V L together forms a single antigen-binding site.
• L light
• H heavy
• immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha ("a"), delta (" ⁇ "), epsilon (“ ⁇ ”), gamma (“ ⁇ ”) and mu (“ ⁇ ”), respectively.
• the ⁇ and a classes are further divided into subclasses (isotypes) on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
• subclasses immunoglobulins
• the subunit structures and three dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al, Cellular and Molecular Immunology, 4 th ed. (W.B. Saunders Co., 2000).
• “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
• V H variable domain
• Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
• an "isolated" molecule or cell is a molecule or a cell that is identified and separated from at least one contaminant molecule or cell with which it is ordinarily associated in the environment in which it was produced.
• the isolated molecule or cell is free of association with all components associated with the production environment.
• the isolated molecule or cell is in a form other than in the form or setting in which it is found in nature. Isolated molecules therefore are distinguished from molecules existing naturally in cells; isolated cells are distinguished from cells existing naturally in tissues, organs, or individuals.
• the isolated molecule is an anti-Clq antibody Fab fragment of the present disclosure.
• the isolated cell is a host cell or hybridoma cell producing an anti-Clq antibody Fab fragment of the present disclosure.
• an "isolated' antibody is one that has been identified, separated and/or recovered from a component of its production environment ⁇ e.g., naturally or recombinantly).
• the isolated polypeptide is free of association with all other contaminant components from its production environment.
• Contaminant components from its production environment such as those resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
• the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N- terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
• Isolated antibody includes the antibody in situ within recombinant T-cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by a process including at least one purification step.
• variable region or “variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody.
• the variable domains of the heavy chain and light chain may be referred to as “V H “ and “V L “, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
• variable refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies.
• the V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
• variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains.
• HVRs hypervariable regions
• the more highly conserved portions of variable domains are called the framework regions (FR).
• the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
• the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies ⁇ see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)).
• the constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent-cellular toxicity.
• CDR complementarity determining region
• CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest” (1991) (also referred to herein as Kabat 1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol.
• CDR-L1 refers, respectively, to the first, second, and third CDRs in a light chain variable region.
• CDR-H1 refers, respectively, to the first, second, and third CDRs in a heavy chain variable region.
• CDR-1 refers, respectively, to the first, second and third CDRs of either chain's variable region.
• monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical except for possible naturally occurring mutations and/or post- translation modifications ⁇ e.g., isomerizations, amidations) that may be present in minor amounts.
• Monoclonal antibodies are highly specific, being directed against a single antigenic site.
• polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
• each monoclonal antibody is directed against a single determinant on the antigen.
• the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
• the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method ⁇ e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2d ed.
• full-length antibody “intact antibody” and “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment.
• whole antibodies include those with heavy and light chains including an Fc region.
• the constant domains may be native sequence constant domains ⁇ e.g., human native sequence constant domains) or amino acid sequence variants thereof.
• the intact antibody may have one or more effector functions.
• antibody fragment comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody.
• antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies ⁇ see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single- chain antibody molecules and multispecific antibodies formed from antibody fragments.
• Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
• the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (C H I).
• V H variable region domain of the H chain
• C H I first constant domain of one heavy chain
• Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen- binding site.
• Pepsin treatment of an antibody yields a single large F(ab') 2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen.
• Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the C H I domain including one or more cysteines from the antibody hinge region.
• Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
• F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
• the Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
• the effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.
• Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions.
• the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
• the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
• composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
• Suitable native-sequence Fc regions for use in the antibodies of the invention include human IgGl, IgG2, IgG3 and IgG4.
• a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
• Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.
• a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s).
• the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide.
• the variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.
• Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
• the preferred FcR is a native sequence human FcR.
• a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
• Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM”) in its cytoplasmic domain.
• Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in its cytoplasmic domain, ⁇ see, e.g., M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
• FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).
• Other FcRs including those to be identified in the future, are encompassed by the term "FcR” herein.
• FcRs can also increase the serum half-life of antibodies.
• Binding to FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having a variant Fc region are administered.
• WO 2004/42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).
• Fv is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
• Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
• the sFv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
• Fully fragments of antibodies comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the F region of an antibody which retains or has modified FcR binding capability.
• antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
• diabodies refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the V H and V L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen- binding sites.
• Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the V H and V L domains of the two antibodies are present on different polypeptide chains.
• Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Nat ⁇ Acad. Sci. USA 90:6444-48 (1993).
• a "chimeric antibody” refers to an antibody (immunoglobulin) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat l Acad. Sci. USA, 81 :6851-55 (1984)).
• Chimeric antibodies of interest herein include PR MATIZED ® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.
• “humanized antibody” is a subset of “chimeric antibodies.”
• Humanized' forms of non-human ⁇ e.g., murine antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
• a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non- human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
• donor antibody such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
• FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
• humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity.
• a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, and the like.
• the number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3.
• the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• a "human antibody is one that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
• Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
• Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice ⁇ see, e.g., U.S. Patent Nos. 6,075,181 and 6, 150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Nat 'l Acad. Sci. USA, 103 :3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
• hypervariable region refers to the regions of an antibody-variable domain that are hypervariable in sequence and/or form structurally defined loops.
• antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
• H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies.
• HVR delineations are in use and are encompassed herein.
• the HVRs that are Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., supra). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
• the AbM HVRs represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody-modeling software.
• the "contact" HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.
• HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (LI), 46-56 or 50- 56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (HI), 50-65 or 49-65 (a preferred embodiment) (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.
• the variable-domain residues are numbered according to Kabat et al., supra, for each of these extended-HVR definitions.
• “Framework” or “ R” residues are those variable-domain residues other than the HVR residues as herein defined.
• a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues ⁇ e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82.
• the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
• the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) ⁇ e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
• the "EU numbering system” or "EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region ⁇ e.g., the EU index reported in Kabat et al, supra).
• the "EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.
• references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system ⁇ e.g., see United States Patent Publication No. 2010-280227).
• acceptor human framework is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework.
• An acceptor human framework "derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer.
• VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
• a "human consensus framework is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
• the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
• the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al, supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.
• amino-acid modification ' ' at a specified position refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. Insertion "adjacent" to a specified residue means insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue.
• the preferred amino acid modification herein is a substitution.
• an "affinity-matured ' ' antibody is one with one or more alterations in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s).
• an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen.
• Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describes affinity maturation by VH- and VL-domain shuffling. Random mutagenesis of HVR and/or framework residues is described by, for example: Barbas et al. Proc Nat. Acad. Sci.
• the term “specifically recognizes ' " or “specifically hinds '” refers to measurable and reproducible interactions such as attraction or binding between a target and an antibody that is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
• an antibody that specifically or preferentially binds to a target or an epitope is an antibody that binds this target or epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets or other epitopes of the target. It is also understood that, for example, an antibody (or a moiety) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
• an antibody that specifically binds to a target may have an association constant of at least about 10 3 M “1 or 10 4 M “1 , sometimes about 10 5 M “1 or 10 6 M “1 , in other instances about 10 6 ⁇ _1 ⁇ 10 7 ⁇ _1 , about 10 8 ⁇ _1 ⁇ 10 9 ⁇ _1 , or about lO ⁇ M ⁇ to 10 11 M "1 or higher.
• a variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
• an "inter action" between a complement protein and a second protein encompasses, without limitation, protein-protein interaction, a physical interaction, a chemical interaction, binding, covalent binding, and ionic binding.
• an antibody “inhibits interaction” between two proteins when the antibody disrupts, reduces, or completely eliminates an interaction between the two proteins.
• An antibody of the present disclosure, or fragment thereof "inhibits interaction” between two proteins when the antibody or fragment thereof binds to one of the two proteins.
• blocking antibody an “antagonist' antibody, an “inhibitory” antibody, or a “neutralizing” antibody is an antibody that inhibits or reduces one or more biological activities of the antigen it binds, such as interactions with one or more proteins.
• blocking antibodies, antagonist antibodies, inhibitory antibodies, or “neutralizing” antibodies substantially or completely inhibit one or more biological activities or interactions of the antigen.
• Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.
• affinity refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen) and is expressed as a dissociation constant (KD).
• Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60- fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1,000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences.
• Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more.
• nM nanomolar
• pM picomolar
• fM femtomolar
• the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.
• the terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and/or antigen-binding fragments.
• binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
• a subject anti-Cls antibody binds specifically to an epitope within a complement Cls protein.
• Specific binding refers to binding with an affinity of at least about 10 "7 M or greater, e.g., 5x lO "7 M, 10 "8 M, 5x l0 "8 M, and greater.
• Non-specific binding refers to binding with an affinity of less than about 10 ⁇ 7 M, e.g., binding with an affinity of 10 "6 M, 10 "5 M, 10 "4 M, etc.
• k on is intended to refer to the rate constant for association of an antibody to an antigen.
• k 0ff is intended to refer to the rate constant for dissociation of an antibody from the antibody/antigen complex.
• K D is intended to refer to the equilibrium dissociation constant of an antibody-antigen interaction.
• percent (%) amino acid sequence identity and “homology” with respect to a peptide, polypeptide or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.
• a “biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay.
• the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
• the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides.
• biological sample encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
• biological sample includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions such as plasma and serum, and the like.
• biological sample also includes solid tissue samples, tissue culture samples, and cellular samples.
• an "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment.
• the isolated nucleic acid molecules encoding the polypeptides and antibodies herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acids encoding any polypeptides and antibodies herein that exist naturally in cells.
• vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• plasmid refers to a circular double stranded DNA into which additional DNA segments may be ligated.
• phage vector refers to a viral vector, wherein additional DNA segments may be ligated into the viral genome.
• viral vector is capable of autonomous replication in a host cell into which they are introduced ⁇ e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
• vectors ⁇ e.g., non-episomal mammalian vectors
• vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
• certain vectors are capable of directing the expression of genes to which they are operatively linked.
• Such vectors are referred to herein as "recombinant expression vectors," or simply, "expression vectors.”
• expression vectors useful in recombinant DNA techniques are often in the form of plasmids.
• plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
• Polynucleotide or “nucleic acid” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.
• the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.
• the sequence of nucleotides may be interrupted by non-nucleotide components.
• a polynucleotide may comprise modification(s) made after synthesis, such as conjugation to a label.
• modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages ⁇ e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages ⁇ e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins ⁇ e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators ⁇ e.g., acridine, psoralen, etc.), those containing chelators ⁇ e.g
• any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports.
• the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
• Other hydroxyls may also be derivatized to standard protecting groups.
• Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside.
• One or more phosphodiester linkages may be replaced by alternative linking groups.
• linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate”), P(S)S ("dithioate”), (0) R 2 ("amidate”), P(0)R, P(0)OR', CO, or CH 2 ("formacetal”), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
• a "host DCr includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts.
• Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
• a host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.
• Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
• physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, di saccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
• buffers such as phosphate, citrate, and other organic acids
• antioxidants including ascorbic acid
• proteins such as serum albumin,
• the methods of the invention provide for modulating the immune response to diseases disclosed herein through administering agents that are antagonists of complement.
• agents that are antagonists of complement.
• immature astrocytes induce expression of Clq proteins in neurons during development.
• Inflammatory mediators such as complement factor are normally expressed at very low levels in healthy brain tissue but can be rapidly induced by a variety of insults to the brain such as infection, ischaemia, and injury.
• Activation of Clq, Clr, and Cls contributes to the inflammatory response, which leads to synaptic loss, along with the generation and recurrence of seizures and seizure-related neuronal damage.
• agents that inhibit complement activation By administering agents that inhibit complement activation, synapses can be maintained that would otherwise be lost.
• agents include an anti-Clq antibody Fab fragment inhibitor, agents that upregulate expression of native complement inhibitors, agents that down-regulate Clq, Clr, or Cls synthesis in neurons, agents that block complement activation, agents that block the signal for complement activation, and the like.
• the methods promote improved maintenance of neuronal function in conditions associated with synapse loss.
• the maintenance of neural connections provides for functional improvement in neurodegenerative disease relative to untreated patients.
• the prevention of synapse loss may comprise at least a measurable improvement relative to a control lacking such treatment over the period of 1, 2, 3, 4, 5, 6 days or at least one week, for example at least a 10% improvement in the number of synapses, at least a 20% improvement, at least a 50% improvement, or more.
• the agents of the present invention are administered at a dosage that decreases synapse loss while minimizing any side-effects. It is contemplated that compositions will be obtained and used under the guidance of a physician for in vivo use.
• the dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like.
• the effective amount of a therapeutic composition to be given to a particular patient will depend on a variety of factors, several of which will be different from patient to patient. Utilizing ordinary skill, the competent clinician will be able to tailor the dosage of a particular therapeutic or imaging composition in the course of routine clinical trials.
• Therapeutic agents e.g., inhibitors of complement, activators of gene expression, etc. can be incorporated into a variety of formulations for therapeutic administration by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
• administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasal, intracheal, etc., administration.
• the active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.
• one strategy for drug delivery through the blood brain barrier entails disruption of the BBB, either by osmotic means such as mannitol or leukotrienes, or biochemically by the use of vasoactive substances such as bradykinin.
• a BBB disrupting agent can be coadministered with the therapeutic compositions of the invention when the compositions are administered by intravascular injection.
• Other strategies to go through the BBB may entail the use of endogenous transport systems, including carrier-mediated transporters such as glucose and amino acid carriers, receptor-mediated transcytosis for insulin or transferrin, and active efflux transporters such as p-glycoprotein.
• Active transport moieties may also be conjugated to the therapeutic or imaging compounds for use in the invention to facilitate transport across the epithelial wall of the blood vessel.
• drug delivery behind the BBB is by intrathecal delivery of therapeutics or imaging agents directly to the cranium, as through an Ommaya reservoir.
• compositions can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human
• the diluent is selected so as not to affect the biological activity of the combination.
• examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution.
• the pharmaceutical composition or formulation can include other carriers, adjuvants, or nontoxic, nontherapeutic, non-immunogenic stabilizers, excipients and the like.
• compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.
• the composition can also include any of a variety of stabilizing agents, such as an antioxidant for example.
• the pharmaceutical composition includes a polypeptide
• the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties ⁇ e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate.
• the polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.
• the pharmaceutical compositions can be administered for prophylactic and/or therapeutic treatments.
• Toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and/or experimental animals, including, for example, determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 5 o.
• Compounds that exhibit large therapeutic indices are preferred.
• the data obtained from cell culture and/or animal studies can be used in formulating a range of dosages for humans.
• the dosage of the active ingredient typically lines within a range of circulating concentrations that include the ED 50 with low toxicity.
• the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
• compositions described herein can be administered in a variety of different ways. Examples include administering a composition containing a
• pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.
• the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
• the active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
• inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
• additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink.
• Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
• Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
• compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process.
• compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.
• compositions of the invention may be administered using any medically appropriate procedure, e.g. intravascular (intravenous, intra-arterial, intracapillary) administration, injection into the cerebrospinal fluid, intracavity or direct injection in the brain.
• Intrathecal administration may be carried out through the use of an Ommaya reservoir, in accordance with known techniques. (F. Balis et al., Am J. Pediatr. Hematol. Oncol. 11, 74, 76 (1989).
• one method for administration of the therapeutic compositions of the invention is by deposition into or near the site by any suitable technique, such as by direct injection (aided by stereotaxic positioning of an injection syringe, if necessary) or by placing the tip of an Ommaya reservoir into a cavity, or cyst, for administration.
• a convection-enhanced delivery catheter may be implanted directly into the site, into a natural or surgically created cyst, or into the normal brain mass.
• Such convection-enhanced pharmaceutical composition delivery devices greatly improve the diffusion of the composition throughout the brain mass.
• the implanted catheters of these delivery devices utilize high-flow microinfusion (with flow rates in the range of about 0.5 to 15.0 ⁇ /minute), rather than diffusive flow, to deliver the therapeutic composition to the brain and/or tumor mass.
• high-flow microinfusion with flow rates in the range of about 0.5 to 15.0 ⁇ /minute
• diffusive flow rather than diffusive flow
• the effective amount of a therapeutic composition to be given to a particular patient will depend on a variety of factors, several of which will be different from patient to patient.
• a competent clinician will be able to determine an effective amount of a therapeutic agent to administer to a patient. Dosage of the agent will depend on the treatment, route of
• compositions can be administered to the subject in a series of more than one administration. For therapeutic compositions, regular periodic administration will sometimes be required, or may be desirable.
• Therapeutic regimens will vary with the agent; for example, some agents may be taken for extended periods of time on a daily or semi-daily basis, while more selective agents may be
• Formulations may be optimized for retention and stabilization in the brain.
• Stabilization techniques include cross-linking, multimerizing, or linking to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc., in order to achieve an increase in molecular weight.
• Implants may be particles, sheets, patches, plaques, fibers, microcapsules and the like and may be of any size or shape compatible with the selected site of insertion.
• the implants may be monolithic, i.e., having the active agent homogenously distributed through the polymeric matrix, or encapsulated, where a reservoir of active agent is encapsulated by the polymeric matrix.
• the selection of the polymeric composition to be employed will vary with the site of administration, the desired period of treatment, patient tolerance, the nature of the disease to be treated and the like. Characteristics of the polymers will include biodegradability at the site of implantation, compatibility with the agent of interest, ease of encapsulation, a half-life in the physiological environment.
• Biodegradable polymeric compositions which may be employed may be organic esters or ethers, which when degraded result in physiologically acceptable degradation products, including the monomers. Anhydrides, amides, orthoesters or the like, by themselves or in combination with other monomers, may find use.
• the polymers will be condensation polymers.
• the polymers may be cross-linked or non-cross-linked. Of particular interest are polymers of hydroxyaliphatic carboxylic acids, either homo- or copolymers, and
• polysaccharides Included among the polyesters of interest are polymers of D-lactic acid, L- lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof.
• L-lactate or D-lactate a slowly biodegrading polymer is achieved, while degradation is substantially enhanced with the racemate.
• Copolymers of glycolic and lactic acid are of particular interest, where the rate of biodegradation is controlled by the ratio of glycolic to lactic acid. The most rapidly degraded copolymer has roughly equal amounts of glycolic and lactic acid, where either homopolymer is more resistant to degradation.
• the ratio of glycolic acid to lactic acid will also affect the brittleness of in the implant, where a more flexible implant is desirable for larger geometries.
• polysaccharides of interest are calcium alginate, and functionalized celluloses, particularly carboxymethylcellulose esters characterized by being water insoluble, a molecular weight of about 5 kD to 500 kD, etc.
• Biodegradable hydrogels may also be employed in the implants of the subject invention. Hydrogels are typically a copolymer material, characterized by the ability to imbibe a liquid. Exemplary biodegradable hydrogels which may be employed are described in Heller in: Hydrogels in Medicine and Pharmacy, N. A. Peppes ed., Vol.
• the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
• a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
• Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• candidate agents are screened for the ability to modulate synapse loss, which agents may include candidate complement inhibitors, variants, fragments, mimetics, agonists and antagonists.
• Such compound screening may be performed using an in vitro model, a genetically altered cell or animal, or purified protein. A wide variety of assays may be used for this purpose.
• compounds that are predicted to be antagonists or agonists of complement including specific complement proteins, e.g., Clq, and complement activating signals, e.g. amyloid, APP, etc. are tested in an in vitro culture system, [as described below.]
• candidate agents may be identified by known pharmacology, by structure analysis, by rational drug design using computer based modeling, by binding assays, and the like.
• Various in vitro models may be used to determine whether a compound binds to, or otherwise affects complement activity.
• candidate compounds are used to contact neurons in an environment permissive for synapse loss.
• Such compounds may be further tested in an in vivo model for an effect on synapse loss.
• Screening may also be performed for molecules produced by astrocytes, e.g., immature astrocytes, which induce Cl q expression in neurons.
• astrocytes e.g., immature astrocytes
• co-cultures of neurons and astrocytes are assessed for the production or expression of molecules that induce Clq expression.
• blocking antibodies may be added to the culture to determine the effect on induction of CI q expression in neurons.
• the neurons are a primary culture, e.g., of retinal ganglion cells (RGCs).
• RGCs retinal ganglion cells
• Purified populations of RGCs are obtained by conventional methods, such as sequential immunopanning.
• the cells are cultured in suitable medium, which will usually comprise appropriate growth factors, e.g., CNTF; BD F; etc.
• the neural cells e.g., RCGs, are cultured for a period of time sufficient allow robust process outgrowth and then cultured with a candidate agent for a period of about 1 day to 1 week.
• the neurons are cultured on a live astrocyte cell feeder in order to induce signaling for synapse loss.
• Methods of culturing astrocyte feeder layers are known in the art.
• cortical glia can be plated in a medium that does not allow neurons to survive, with removal of nonadherent cells.
• cultures are fixed, blocked and washed, then stained with antibodies specific synaptic proteins, e.g., synaptotagmin, etc. and visualized with an appropriate reagent, as known in the art. Analysis of the staining may be performed microscopically.
• digital images of the fluorescence emission are with a camera and image capture software, adjusted to remove unused portions of the pixel value range and the used pixel values adjusted to utilize the entire pixel value range.
• Corresponding channel images may be merged to create a color (RGB) image containing the two single-channel images as individual color channels.
• Co-localized puncta can be identified using a rolling ball background subtraction algorithm to remove low-frequency background from each image channel. Number, mean area, mean minimum and maximum pixel intensities, and mean pixel intensities for all synaptotagmin, PSD-95, and colocalized puncta in the image are recorded and saved to disk for analysis.
• agent as used herein describes any molecule, e.g., protein or
• Candidate agents also include genetic elements, e.g., anti-sense and RNAi molecules to inhibit Clq expression, and constructs encoding complement inhibitors, e.g., CD 59, and the like.
• Candidate agents encompass numerous chemical classes, though typically they are organic molecules, including small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
• Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
• the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
• Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
• a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
• one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
• neurological or “cognitive” function as used herein, it is meant that the decrease of synapses in the brain enhances the patient's ability to think, function, etc.
• subject encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;
• laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
• the term does not denote a particular age or gender.
• neurodegenerative conditions e.g., Alzheimer's disease, Down syndrome, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, myotonic dystrophy, glaucoma, Parkinson's disease; and the like.
• complement-associated eye conditions such as choroidal neovascularization (CNV) and age-related macular degeneration (AMD).
• CNV choroidal neovascularization
• AMD age-related macular degeneration
• synaptogenesis such as thrombospondins
• thrombospondins may also be administered.
• autoimmune conditions or complement-mediated eye conditions.
• Age-related Macular Degeneration is the leading cause of blindness in the elderly worldwide.
• AMD is characterized by a progressive loss of central vision attributable to degenerative and neovascular changes in the macula, a highly specialized region of the ocular retina responsible for fine visual acuity.
• Recent estimates indicate that 14 million persons are blind or severely visually impaired because of AMD.
• the disease has a tremendous impact on the physical and mental health of the geriatric population and their families and is becoming a major public health burden. New discoveries, however, are beginning to provide a clearer picture of the relevant cellular events, genetic factors, and biochemical processes associated with early AMD and how AMD is a complement- associated eye condition.
• nonexudative AMD Two types exist, non-exudative (dry) and exudative (wet) AMD.
• the dry, or nonexudative, form involves atrophic and hypertrophic changes in the retinal pigment epithelium (RPE) underlying the central retina (macula) as well as deposits (drusen) on the RPE.
• RPE retinal pigment epithelium
• drusen deposits
• Patients with nonexudative AMD can progress to the wet, or exudative, form of AMD, in which abnormal blood vessels called choroidal neovascular membranes (CNVMs) develop under the retina, leak fluid and blood, and ultimately cause a blinding disciform scar in and under the retina.
• CNVMs choroidal neovascular membranes
• Nonexudative AMD which is usually a precursor of exudative AMD, is more common.
• the presentation of nonexudative AMD varies; hard drusen, soft drusen, RPE geographic at
• Complement components are deposited on the RPE early in AMD and are major constituents of drusen.
• Alzheimer's disease is a progressive, inexorable loss of cognitive function associated with an excessive number of senile plaques in the cerebral cortex and subcortical gray matter, which also contains—amyloid and neurofibrillary tangles consisting of tau protein.
• the common form affects persons > 60 yr old, and its incidence increases as age advances. It accounts for more than 65% of the dementias in the elderly.
• Alzheimer's disease The cause of Alzheimer's disease is not known. The disease runs in families in about 15 to 20% of cases. The remaining, so-called sporadic cases have some genetic determinants. The disease has an autosomal dominant genetic pattern in most early-onset and some late-onset cases but a variable late-life penetrance. Environmental factors are the focus of active investigation.
• Cerebral glucose use and perfusion is reduced in some areas of the brain (parietal lobe and temporal cortices in early-stage disease, prefrontal cortex in late-stage disease).
• Neuritic or senile plaques (composed of neurites, astrocytes, and glial cells around an amyloid core) and neurofibrillary tangles (composed of paired helical filaments) play a role in the pathogenesis of Alzheimer's disease.
• Senile plaques and neurofibrillary tangles occur with normal aging, but they are much more prevalent in persons with Alzheimer's disease.
• Amyotrophic lateral sclerosis is a rapidly progressive, invariably fatal neurological disease that attacks motor neurons. Muscular weakness and atrophy and signs of anterior horn cell dysfunction are initially noted most often in the hands and less often in the feet. The site of onset is random, and progression is asymmetric. Cramps are common and may precede weakness. Rarely, a patient survives 30 yr; 50% die within 3 yr of onset, 20%) live 5 yr, and 10%> live 10 yr. Diagnostic features include onset during middle or late adult life and progressive, generalized motor involvement without sensory abnormalities. Nerve conduction velocities are normal until late in the disease.
• Down Syndrome is a chromosomal disorder usually resulting in mental retardation, a characteristic facies, and many other typical features, including microcephaly and short stature. In about 95% of cases, there is an extra whole chromosome 21. At autopsy, adult Down syndrome brains show the typical microscopic findings of Alzheimer's disease, and many persons also develop the associated clinical signs.
• Parkinson's Disease is an idiopathic, slowly progressive, degenerative CNS disorder characterized by slow and decreased movement, muscular rigidity, resting tremor, and postural instability.
• primary Parkinson's disease the pigmented neurons of the substantia nigra, locus caeruleus, and other brain stem dopaminergic cell groups are lost. The cause is not known.
• Secondary parkinsonism results from loss of or interference with the action of dopamine in the basal ganglia due to other idiopathic degenerative diseases, drugs, or exogenous toxins.
• the most common cause of secondary parkinsonism is ingestion of antipsychotic drugs or reserpine, which produce parkinsonism by blocking dopamine receptors.
• Less common causes include carbon monoxide or manganese poisoning, hydrocephalus, structural lesions (tumors, infarcts affecting the midbrain or basal ganglia), subdural hematoma, and degenerative disorders, including striatonigral degeneration.
• Myotonic dystrophy is an autosomal dominant multisystem disorder characterized by dystrophic muscle weakness and myotonia.
• the molecular defect is an expanded trinucleotide (CTG) repeat in the 3' untranslated region of the myotonin-protein kinase gene on chromosome 19q.
• CCG trinucleotide
• Symptoms can occur at any age, and the range of clinical severity is broad. Myotonia is prominent in the hand muscles, and ptosis is common even in mild cases. In severe cases, marked peripheral muscular weakness occurs, often with cataracts, premature balding, hatchet facies, cardiac arrhythmias, testicular atrophy, and endocrine abnormalities (e.g., diabetes mellitus). Mental retardation is common. Severely affected persons die by their early 50s.
• Glaucoma is a common neurodegenerative disease that affects retinal ganglion cells (RGCs). Substantial effort is being expended to determine how RGCs die in glaucoma. Evidence supports the existence of compartmentalized degeneration programs in synapses and dendrites, including RGCs. Recent data, from in vitro studies and from an inherited mouse model of glaucoma, suggest that molecularly distinct degenerative pathways underlie the destruction of RGC somata and RGC axons. In various neurodegenerative diseases, axons, dendrites and synapses often degenerate well before the cells die, and there is increasing evidence that this is important for the production of clinical symptoms and signs.
• Huntington's disease is a hereditary progressive neurodegenerative disorder characterized by the development of emotional, behavioral, and psychiatric abnormalities; loss of previously acquired intellectual or cognitive functioning; and movement abnormalities (motor disturbances).
• the classic signs of HD include the development of chorea - or involuntary, rapid, irregular, jerky movements that may affect the face, arms, legs, or trunk - as well as the gradual loss of thought processing and acquired intellectual abilities
• HD is transmitted within families as an autosomal dominant trait.
• the disorder occurs as the result of abnormally long sequences or "repeats" of coded instructions within a gene on chromosome 4 (4pl6.3).
• the progressive loss of nervous system function associated with HD results from loss of neurons in certain areas of the brain, including the basal ganglia and cerebral cortex.
• Multiple Sclerosis is characterized by various symptoms and signs of CNS dysfunction, with remissions and recurring exacerbations.
• the most common presenting symptoms are paresthesias in one or more extremities, in the trunk, or on one side of the face; weakness or clumsiness of a leg or hand; or visual disturbances, e.g., partial blindness and pain in one eye (retrobulbar optic neuritis), dimness of vision, or scotomas.
• the methods of the invention can find use in combination with cell or tissue transplantation to the central nervous system, where such grafts include neural progenitors such as those found in fetal tissues, neural stem cells, embryonic stem cells or other cells and tissues contemplated for neural repair or augmentation.
• neural progenitors such as those found in fetal tissues, neural stem cells, embryonic stem cells or other cells and tissues contemplated for neural repair or augmentation.
• Neural stem/progenitor cells have been described in the art, and their use in a variety of therapeutic protocols has been widely discussed. For example, inter alia, U.S. Patent nos. 6,638,501, Bjornson et al.; U.S.
• Neural stem and progenitor cells can participate in aspects of normal development, including migration along well-established migratory pathways to disseminated CNS regions, differentiation into multiple developmentally- and regionally-appropriate cell types in response to microenvironmental cues, and non-disruptive, non-tumorigenic interspersion with host progenitors and their progeny.
• Human NSCs are capable of expressing foreign transgenes in vivo in these disseminated locations. A such, these cells find use in the treatment of a variety of conditions, including traumatic injury to the spinal cord, brain, and peripheral nervous system; treatment of degenerative disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease; affective disorders including major depression; stroke; and the like.
• synapse loss enhancers the functional connections of the neurons are enhances, providing for an improved clinical outcome.
• compositions The antibody Fab fragments of the present invention may be administered for the treatment of complement-associates eye conditions in the form of pharmaceutical
• Therapeutic formulations of an antibody Fab fragment of the invention are prepared for storage by mixing the antibody Fab fragment having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers ⁇ Remington's
• Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
• hexamethonium chloride benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;
• polypeptides such as serum albumin, gelatin, or immunoglobulins
• hydrophilic polymers such as polyvinylpyrrolidone
• amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine
• chelating agents such as EDTA
• sugars such as sucrose, mannitol, trehalose or sorbitol
• salt-forming counter-ions such as sodium
• metal complexes e.g. Zn-protein complexes
• non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
• Lipofections or liposomes can also be used to deliver the antibody Fab fragment into cell, wherein the smallest fragment which specifically binds to the binding domain of the target protein is preferred.
• the antibody Fab fragment may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)
• microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in
• sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody Fab fragment, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
• sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
• copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
• the compounds of the invention for prevention or treatment of an ocular disease or condition are typically administered by ocular, intraocular, and/or intravitreal injection.
• Other methods administration by also be used, which includes but is not limited to, topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intralesional administration.
• Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.
• Formulations for ocular, intraocular or intravitreal administration can be prepared by methods and using ingredients known in the art.
• a main requirement for efficient treatment is proper penetration through the eye. Unlike diseases of the front of the eye, where drugs can be delivered topically, retinal diseases require a more site-specific approach. Eye drops and ointments rarely penetrate the back of the eye, and the blood-ocular barrier hinders penetration of systemically administered drugs into ocular tissue. Accordingly, usually the method of choice for drug delivery to treat retinal disease, such as AMD and CNV, is direct intravitreal injection. Intravitreal injections are usually repeated at intervals which depend on the patient's condition, and the properties and half-life of the drug delivered. For intraocular (e.g., intravitreal) penetration, usually molecules of smaller size are preferred.
• the efficacy of the treatment of complement-associated eye conditions can be measured by various endpoints commonly used in evaluating intraocular diseases.
• vision loss can be assessed.
• Vision loss can be evaluated by, but not limited to, e.g., measuring by the mean change in best correction visual acuity (BCVA) from baseline to a desired time point (e.g., where the BCVA is based on Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart and assessment at a test distance of 4 meters), measuring the proportion of subjects who lose fewer than 15 letters in visual acuity at a desired time point compared to baseline, measuring the proportion of subjects who gain greater than or equal to 15 letters in visual acuity at a desired time point compared to baseline, measuring the proportion of subjects with a visual-acuity Snellen equivalent of 20/2000 or worse at a desired time point, measuring the EI Visual
• BCVA mean change in best correction visual acuity
• EDRS Early Treatment Diabetic Retinopathy Study
• Ocular assessments can be done, e.g., which include, but are not limited to, e.g., performing eye exam, measuring intraocular pressure, assessing visual acuity, measuring slitlamp pressure, assessing intraocular inflammation, etc.
• Antibody Fab fragments of the present disclosure may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567.
• isolated nucleic acids having a nucleotide sequence encoding any of the antibody Fab fragments of the present disclosure are provided. Such nucleic acids may encode an amino acid sequence containing the V L /C L and/or an amino acid sequence containing the V H /C H 1 of the anti-Clq antibody.
• one or more vectors ⁇ e.g., expression vectors) containing such nucleic acids are provided.
• a host cell containing such nucleic acid is also provided.
• the host cell contains ⁇ e.g., has been transduced with): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing the V L /C L of the antibody and an amino acid sequence containing the V H /C H 1 of the antibody, or (2) a first vector containing a nucleic acid that encodes an amino acid sequence containing the V L /C L of the antibody and a second vector containing a nucleic acid that encodes an amino acid sequence containing the V H /C H 1 of the antibody.
• the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell ⁇ e.g., Y0, NS0, Sp20 cell).
• the method includes culturing a host cell of the present disclosure containing a nucleic acid encoding the anti-Clq antibody Fab fragment, under conditions suitable for expression of the antibody Fab fragment.
• the antibody Fab fragment is subsequently recovered from the host cell (or host cell culture medium).
• a nucleic acid encoding the anti-Clq antibody Fab fragment is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
• Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
• Suitable vectors containing a nucleic acid sequence encoding any of the anti-Clq antibody Fab fragment of the present disclosure, or fragments thereof polypeptides (including antibodies) described herein include, without limitation, cloning vectors and expression vectors.
• Suitable cloning vectors can be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
• Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
• Bluescript e.g., pBS SK+
• mpl8 mpl9 mpl9
• pBR322 mpl9
• ColEl ColEl
• pCRl pCRl
• RP4 phage DNAs
• shuttle vectors such as pSA3 and pAT28.
• Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure.
• the expression vector may replicable in the host cells either as episomes or as an integral part of the chromosomal DNA.
• Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno- associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT
• Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.
• the vectors containing the nucleic acids of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus).
• electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances
• microprojectile bombardment e.g., where the vector is an infectious agent such as vaccinia virus.
• infection e.g., where the vector is an infectious agent such as vaccinia virus.
• the vector contains a nucleic acid containing one or more amino acid sequences encoding an anti- Clq antibody of the present disclosure.
• Suitable host cells for cloning or expression of antibody Fab fragment-encoding vectors include prokaryotic or eukaryotic cells.
• anti-Clq antibody Fab fragments of the present disclosure may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
• For expression of antibody fragments and polypeptides in bacteria e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli).
• the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
• eukaryotic microorganisms such as filamentous fungi or yeast
• suitable cloning or expression hosts for antibody-encoding vectors including fungi and yeast strains whose glycosylation pathways have been "humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern (e.g., Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006)).
• Suitable host cells for the expression of glycosylated antibody Fab fragments can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts (e.g., U.S. Patent Nos. 5,959, 177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, describing
• Vertebrate cells may also be used as hosts.
• mammalian cell lines that are adapted to grow in suspension may be useful.
• Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
• monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et ⁇ ., Annals N.Y. Acad. Sci. 383 :44-68 (1982); MRC 5 cells; and FS4 cells.
• Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
• complement inhibitors of the present disclosure may be used, without limitation, conjointly with any additional treatment, such as immunosuppressive therapies, for any disease disclosed herein, including autoimmune and/or neurodegenerative diseases.
• the antibodies of this disclosure are administered in combination with an inhibitor of the alternative pathway of complement activation.
• inhibitors may include, without limitation, factor B blocking antibodies, factor D blocking antibodies, soluble, membrane-bound, tagged or fusion-protein forms of CD59, DAF, CR1, CR2, Crry or Comstatin-like peptides that block the cleavage of C3, non-peptide C3aR antagonists such as SB 290157, Cobra venom factor or non-specific complement inhibitors such as nafamostat mesilate (FUTHAN; FUT-175), aprotinin, K-76 monocarboxylic acid (MX-1) and heparin ⁇ see, e.g., T.E.
• the antibodies of this disclosure are administered in combination with an inhibitor of the interaction between the autoantibody and its autoantigen.
• inhibitors may include purified soluble forms of the autoantigen, or antigen mimetics such as peptide or RNA-derived mimotopes, including mimotopes of the AQP4 antigen.
• inhibitors may include blocking agents that recognize the autoantigen and prevent binding of the autoantibody without triggering the classical complement pathway.
• blocking agents may include, e.g., autoantigen-binding RNA aptamers or antibodies lacking functional Clq binding sites in their Fc domains ⁇ e.g., Fab fragments or antibody otherwise engineered not to bind Clq).
• kits containing antibody Fab fragments of this disclosure include one or more containers comprising a purified anti-Clq antibody Fab fragment and instructions for use in accordance with methods known in the art.
• these instructions comprise a description of administration of the inhibitor to treat or diagnose a disease, according to any methods known in the art.
• the kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the disease and the stage of the disease.
• the instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
• the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
• Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
• the label or package insert indicates that the composition is used for treating a specific disease. Instructions may be provided for practicing any of the methods described herein.
• kits of this invention are in suitable packaging.
• suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
• packages for use in combination with a specific device such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
• a kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
• the container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
• At least one active agent in the composition is an inhibitor of classical complement pathway.
• the container may further comprise a second pharmaceutically active agent.
• Kits may optionally provide additional components such as buffers and interpretive information.
• the kit comprises a container and a label or package insert(s) on or associated with the container.
• a direct ELISA was used to assess binding of Fab and full-length antibodies to human Clq (Figure 1). Briefly, black 96-well plates (#3925, Corning) were coated with human Clq (2 ug/mL) (Complement Technology) in Bicarbonate buffer (pH 9.4) (Thermo Scientific), overnight at 4C. Next day the plates are washed with dPBS thrice and blocked with 3% BSA/PBS for 1 hour at room temperature (RT). Serial dilutions of Ml, Ml-Fab were prepared in dPBS 0.3% BSA, 0.1% Tween buffer (50 uL/well).
• Luminescence counts were measured using Envision plate reader (Perkin Elmer). Counts are plotted as a function of concentration and a 4-parameter logistic fit was used to derive EC50 for Clq binding. As demonstrated in Figure 1, the Ml Fab has the same binding affinity as Ml whole antibody. Figure IB shows Ml whole antibody (bivalent) and Ml Fab
• antibodies (Ml-whole IgG, Ml-Fab, Humanized Ml and Humanized Ml-Fab) were titrated over a range of 10000 to 10 ng/mL in 50 x diluted normal human serum in GVB++ buffer (Complement Technology). Then, 50 uL of diluted serum was mixed with 50 uL of diluted sheep RBCs (-100 million cells/mL) (Complement Technology). The samples were incubated at 37C for 30 minutes. Then the plates were centrifuged at 2000 rpm for 5 minutes, supernatant collected and absorbance read at 415 nm. Controls were run that included - background hemolysis in GVB++ buffer alone without serum and maximal hemolysis triggered using water.
• Ml Fab has the same functional potency as Ml whole antibody. It was anticipated that Ml whole antibody would exhibit higher potency because of its ability to engage multiple functional head groups of Clq ( Figure IB), and because of the larger size for stearic hindrance of other head groups.

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