FAB fragment of anti-complement factor C1Q and its application

Abstract

The present invention relates to FAB fragments of anti-complement factor C1q and their applications. The present invention generally discloses anti-C1q antibody Fab fragments, host cells expressing antibody Fab fragments, DNA sequences encoding heavy chain regions and light chain regions of antibody Fab fragments, and cloning vectors or expression vectors containing the DNA sequences, methods for producing antibody Fab fragments, pharmaceutical compositions comprising antibody Fab fragments, applications of antibody Fab fragments in the manufacture of drugs for inhibiting synaptic loss in patients with adverse synaptic loss, treating diseases associated with complement activation in individuals in need and reducing the risk of developing diseases associated with complement activation in subjects, and applications of antibody Fab fragments in the preparation of kits for determining the risk of developing diseases associated with complement activation in subjects.

Description

[0001] This application is a divisional application of the PCT international application PCT / US2016 / 063587 filed on November 23, 2016, which entered the Chinese national phase on July 20, 2018, and whose Chinese patent application number is 201680079670.4 and whose invention name is "FAB fragment of anti-complement factor C1Q and its application".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 259,227, filed on November 24, 2015, which is hereby incorporated by reference in its entirety. Technical Field

[0004] The present invention relates to a FAB fragment of anti-complement factor C1Q and application thereof. Background Art

[0005] Acute or chronic inflammation is a common component of many clinical disorders, and the complement system has been associated with an increasing number of inflammatory conditions including degenerative diseases, cancer and transplant rejection. The complement system acts as a sensor for pathogens, identifies pathological and damaged host cells, and closely collaborates with other immune systems and defense systems to eliminate potential dangers. However, insufficient, excessive or poorly controlled complement activation can tilt the balance between health and disease, and cause self-attack on host cells. Such immune imbalance can stimulate the circulation between complement, inflammatory cells and tissue damage, which regenerates inflammatory stimuli rather than eliminating them and worsening clinical complications. The inappropriate activation of complement has been linked to many autoimmune diseases, inflammatory diseases and neurodegenerative diseases, as well as ischemia-reperfusion injury and cancer. Therefore, the therapeutic regulation of complement activity emerges as an attractive target for upstream inhibition of inflammatory processes. Summary of the invention

[0006] The present disclosure is generally directed to anti-C1q Fab fragments and uses thereof.

[0007] Complement is a major component of the innate immune system, particularly because it is associated with inflammation and the body's defense against invading organisms. Complement is also involved in the removal of autoantigens and apoptotic cells, forming a bridge to adaptive immunity, and also plays an important role in tissue regeneration and tumor growth. In order to perform these functions, the complement system relies on the interaction of soluble proteins and cell surface-bound proteins, which interact with the pathogen cell surface to mark them for destruction by phagocytes. The complement system is composed of a large number of different plasma proteins produced mainly by the liver. Some of these proteins are a class of proteases referred to as zymogens, which are themselves activated by proteolytic cleavage. These zymogens can be widely distributed without being in activity until local pathogens are detected. Therefore, the complement system is activated by the enzyme cascade reaction triggered.

[0008] Complement activation is triggered through three pathways: the classical pathway, the alternative pathway, and the lectin pathway. All three pathways are based on the detection of surface structures by pattern recognition proteins. In addition, all three pathways converge through a common intersection (complement C3). C3 is an acute phase reactant. Although a small amount is also produced by activated monocytes and macrophages, the liver is the main site of synthesis. A single-chain precursor (pro-C3) of approximately 200 kD is found in the cell; cDNA shows that it includes 1663 amino acids. This is processed into α subunits and β subunits by proteolytic cleavage, and in the mature protein, the α subunit and the β subunit are connected by a disulfide bond. Pro-C3 contains a signal peptide of 22 amino acid residues, a β chain (645 residues) and an α chain (992 residues). The two chains are connected by 4 arginine residues (not present in the mature protein).

[0009] The classical pathway is activated by direct binding of the complement protein C1q to plaques of surface-bound antibodies (IgM and IgG), as well as to C-reactive protein, serum amyloid P, pentraxin 3 and other ligands on the surface of apoptotic or microbial cells.

[0010] C1q is a large multimeric protein of 460 kDa, consisting of 18 polypeptide chains (6 C1q A chains, 6 C1q B chains and 6 C1q C chains). The C1r and C1s complement proteins bind to the C1q tail region to form the C1q complex. Binding of the C1q complex to the surface of the cell or to the complement binding domain of the Fc region of an antibody induces a conformational change in C1q that leads to activation of the autocatalytic enzymatic activity in C1r, which then cleaves C1s to generate an active serine protease. Once activated, C1s cleaves C4, etc., leading to a complement cascade sequence. Ultimately this pathway leads to the formation of a membrane attack complex that lyses and kills infected cells.

[0011] Since complement can attack both foreign invaders and host cells, complement is nonspecific. Under normal conditions, host cells, including neurons, are protected from potential complement-mediated damage by various mobile phases and membrane-bound complement regulatory proteins such as C1 inhibitor (C1-Inh). C1-INH dissociates C1r and C1s from the active C1 complex, protecting 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. However, the lack of these components or the overactivation of complement in response to certain pathological states can defeat this protective mechanism. Such unbalanced activation has been associated with an increasing number of diseases and pathological disorders.

[0012] For example, various complement components are expressed in vitro and in vivo by neurons and glial cells. Although their functions in the brain are unknown, the expression of many of these complement proteins is upregulated by serum or inflammatory cytokines after brain injury or during the pathological process of neurodegenerative diseases. Astrocytes in the culture medium have been reported to express C1q, C1r, C1s, C4, C2 and C3, as well as more terminal proteins. Neurons have been reported to express C4 and C3. C1q has been shown to be expressed in neuronal synapses, and marks these synapses for removal. See, for example, U.S. Patent Publication Nos. 2012 / 0195880 and 2012 / 328601. Although selective synaptic loss is an essential aspect of normal brain development ("synaptic pruning"), excessive synaptic loss, particularly in mature or aging brains, causes neurodegeneration and cognitive decline. Elevated synaptic complement expression has been found to contribute to synaptic loss in normal aging and the progression of neurodegenerative diseases. In contrast, reducing neuronal complement expression was found to be neuroprotective. Neurons affected by synaptic loss can be central nervous system neurons, or peripheral system neurons. Based on these findings, neutralizing the activity of complement factors (such as C1q) is considered to be a promising therapeutic strategy for preventing synaptic loss and slowing the progression of neurodegenerative diseases and cognitive decline in normal aging. Neurodegenerative diseases that involve synaptic loss and are considered to withstand treatment with the neutralization of complement factors (such as C1q) as the target include Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Down syndrome, Parkinson's disease, Huntington's disease, etc.

[0013] In addition, the anaphylatoxin C5a has been shown to contribute to tumor growth in mice. In these cases where complement has toxic effects, it is desirable to regulate the activation of complement by using appropriate complement inhibitors. Currently, most complement inhibitors are immunogenic protein drugs and have poor tissue penetration. For example, the failure of the anti-C5 mAb pexelizumab (Alexion Pharmaceuticals) to treat acute myocardial infarction may be caused in part by its poor tissue penetration. Unlike protein inhibitors, several low molecular weight drugs (less than 2 kDa) have been used as complement inhibitors and do not have the same disadvantages as protein inhibitors. However, many of the low molecular weight drugs and other small molecule inhibitors that have been reported (such as bisphenol pyrosulfate, steroids and triterpenoids) generally have low efficacy against complement. In addition, low molecular weight and small molecule complement inhibitors have other problems, including poor selectivity and high toxicity. Another challenge for low molecular weight and small molecule complement inhibitors is that the complement cascade depends on a large number of protein-protein interactions, so compared to, for example, the pocket of an enzyme, the interaction surface is usually much larger, and the amino acid residues involved in such interactions are usually not adjacent. In addition, the contact surface is usually shallow and lacks any groove that enables small compounds to be tightly bound. Therefore, it illustrates that all physiological complement regulators (including protease inhibitor C1-Inh) are relatively large proteins. Despite the challenges of the above-mentioned protein-drugs, low molecular weight and small molecule complement inhibitors, antibody fragments (Fab) for the inhibition of different components of the complement cascade can be advantageous for preventive, diagnostic and therapeutic clinical applications. In the case of rapid clearance, higher tissue penetration ability, lack of Fc-mediated effector functions (such as CDC, ADCC and phagocytosis) and lower immunogenicity, such Fab molecules are desirable. In addition, Fab molecules do not form cross-linked immune complexes that can trigger allergic reactions.

[0014] In certain aspects, the disclosure provides antibody Fab fragments that bind to proteins in the complement cascade. In some embodiments, the antibody Fab fragment binds to the C1q protein. In some embodiments, the antibody Fab fragment comprises a heavy chain and a light chain, wherein the heavy chain is truncated after the first heavy chain domain.

[0015] In a preferred embodiment, the antibody Fab fragment is an anti-C1q antibody Fab fragment.

[0016] In one aspect, the disclosure provides antibody Fab fragments that bind to CIq.

[0017] In some embodiments, the antibody Fab fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises SEQ ID NO:1 and the light chain variable domain comprises SEQ ID NO:3.

[0018] In one aspect, the present disclosure provides an antibody Fab, wherein the antibody Fab fragment comprises complementarity determining regions (CDRs) of the heavy chain and the light chain, and wherein

[0019] The heavy chain CDR1 sequence includes amino acid residues 26-35 of SEQ ID NO: 1, the heavy chain CDR2 sequence includes amino acid residues 50-66 of SEQ ID NO: 1, and the heavy chain CDR3 sequence includes amino acid residues 99-110 of SEQ ID NO: 1, and

[0020] The light chain CDR1 sequence includes amino acid residues 24-34 of SEQ ID NO:3, the light chain CDR2 sequence includes amino acid residues 50-56 of SEQ ID NO:3, and the light chain CDR3 sequence includes amino acid residues 89-97 of SEQ ID NO:3.

[0021] In one aspect, the present disclosure provides an antibody Fab fragment that binds to a protein in the complement cascade, wherein the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 5-8, or an amino acid sequence having at least 95% homology to an amino acid sequence from SEQ ID NOs: 5-8.

[0022] In some embodiments, the light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 9-12, or an amino acid sequence having at least 90% homology to an amino acid sequence from SEQ ID NOs: 9-12.

[0023] In some embodiments, antibody Fab fragment can be prepared by any suitable method known in the art.For example, any suitable enzymatic cleavage and / or enzyme digestion technology can be used to obtain antibody Fab fragment from any full antibody (especially full monoclonal antibody), such as with cysteine ​​protease papain treatment to produce antibody Fab fragment.

[0024] In one embodiment, the antibody Fab fragments of the present invention are prepared by using recombinant DNA technology involving manipulation and re-expression of DNA encoding the variable and constant regions of the antibody. Standard molecular biology techniques can be used to modify, add or delete additional amino acids or domains as desired. Any changes to the variable or constant regions are still included by the terms "variable" and "constant" regions as used herein. Preferably, PCR is used immediately following the C H 1 introduces a stop codon, making C HThe translation of the 1 domain is terminated at the interchain cysteine. Methods for designing suitable PCR primers are well known in the art, and antibody C H The sequence of the 1 domain is readily available. In some embodiments, a stop codon can be introduced using site-directed mutagenesis techniques.

[0025] In some cases, the antibody Fab fragment starting material of the present invention can be derived from any antibody isotype ("class") (including, for example, IgG, IgM, IgA, IgD and IgE) and its subclasses (including, for example, IgG1, IgG2, IgG3 and IgG4). Preferably, the antibody Fab fragment of the present invention is derived from IgG1.

[0026] In some embodiments, antibody Fab fragment starting material can be obtained from any species (including, for example, mouse, rat, rabbit, pig, hamster, camel, llama, goat or human). In a preferred embodiment, the heavy chain and light chain of the antibody Fab fragment are from murine IgG1. The portion of the antibody Fab fragment can be obtained from more than one species. For example, the antibody fragment can be chimeric or humanized. In one example, the constant region is from one species, and the variable region is from another species. In another example, the antibody Fab fragment is humanized.

[0027] The antibody fragment starting material also can be modified.In one example, the variable region of antibody fragment has been produced using recombinant DNA engineering technology.Such engineered versions include those produced from the variable region of natural antibodies by, for example, insertion, deletion or change of the amino acid sequence of natural antibodies.The specific examples of this type include those engineered variable regions containing at least one CDR from an antibody and optional one or more framework amino acids and the residual part from the variable region of a second antibody.

[0028] In some embodiments, 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. In some embodiments, a DNA sequence encoding the amino acids of SEQ ID NOs 1 and 2 is provided. In one embodiment, the nucleic acid sequence encoding SEQ ID NO: 1 is SEQ ID NO: 3. In another embodiment, the nucleic acid sequence encoding SEQ ID NO: 2 is SEQ ID NO: 4.

[0029] Also disclosed are antibody Fab fragments, wherein the heavy chain and the light chain comprise sequences having at least 90% identity or similarity to the sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Preferably, 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.

[0030] In some embodiments, the antibody Fab fragment specifically binds to human C1q, mouse C1q, dog C1q, rhesus monkey C1q, cynomolgus monkey C1q, or rat C1q. In some embodiments, the antibody Fab fragment specifically binds to both human C1q and mouse C1q. In some embodiments, the antibody Fab fragment specifically binds to both human C1q and rat C1q. In some embodiments, the antibody Fab fragment specifically binds to both human C1q and dog C1q. In some embodiments, the antibody Fab fragment specifically binds to both human C1q and rhesus monkey C1q. In some embodiments, the antibody Fab fragment specifically binds to both human C1q and cynomolgus monkey C1q. In some embodiments, the antibody Fab fragment specifically binds to both mouse C1q and rat C1q. In some embodiments, the antibody Fab fragment specifically binds to both mouse C1q and dog C1q. In some embodiments, the antibody Fab fragment specifically binds to both mouse C1q and rhesus monkey C1q. In some embodiments, the antibody Fab fragment specifically binds to both mouse C1q and cynomolgus monkey C1q. In some embodiments, the antibody Fab fragment specifically binds to both rat C1q and dog C1q. In some embodiments, the antibody Fab fragment specifically binds to both rat C1q and rhesus monkey C1q. In some embodiments, the antibody Fab fragment specifically binds to both rat C1q and cynomolgus monkey C1q. In some embodiments, the antibody Fab fragment specifically binds to both dog C1q and rhesus monkey C1q. In some embodiments, the antibody Fab fragment specifically binds to both dog C1q and cynomolgus monkey C1q. In some embodiments, the antibody Fab fragment specifically binds to rhesus monkey dog ​​C1q and cynomolgus monkey C1q. In some embodiments, the antibody Fab fragment specifically binds to human C1q, mouse C1q and / or rat C1q, and at least one of the following: dog C1q, rhesus monkey C1q, and cynomolgus monkey C1q.

[0031] In some embodiments, the antibody Fab fragment binds to both human CIq and mouse CIq. In other embodiments, the antibody Fab fragment binds to human CIq, mouse CIq, rat CIq, dog CIq, rhesus monkey CIq, and cynomolgus monkey CIq.

[0032] In one aspect, the present disclosure provides an antibody Fab fragment that specifically binds to a C1q protein, the antibody Fab fragment comprising:

[0033] a) a heavy chain variable domain, the heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 5-8, or an amino acid sequence having at least 95% homology to an amino acid sequence from SEQ ID NOs: 5-8; and / or

[0034] b) a light chain variable domain, the light chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 9-12, or an amino acid sequence having at least 90% homology to an amino acid sequence from SEQ ID NOs: 9-12.

[0035] In some embodiments, the antibody Fab fragment binds to substantially the same C1q epitope as antibody M1 produced by a hybridoma cell line having ATCC Accession No. PTA- 120399. In some cases, the antibody Fab fragment inhibits the binding of monoclonal antibody M1 produced by a hybridoma cell line having ATCC Accession No. PTA-120399 to human C1q or to mouse C1q.

[0036] In some embodiments, the antibody Fab fragment specifically binds to both human C1q and mouse C1q. In some embodiments, the antibody Fab fragment binds to rat C1q, dog C1q, rhesus monkey C1q, or cynomolgus monkey C1q, or a combination thereof. In some embodiments, the antibody Fab fragment binds to human C1q, mouse C1q, and / or rat C1q, and at least one of the following: dog C1q, rhesus monkey C1q, and cynomolgus monkey C1q.

[0037] In some embodiments, the antibody Fab fragment is of the IgG class. In some embodiments, the IgG class is IgG1.

[0038] In some embodiments, provided herein is a humanized anti-C1q antibody Fab fragment that binds to an epitope of C1q that is identical or overlapping with an epitope of C1q bound by another antibody of the present disclosure. In certain embodiments, the humanized anti-C1q antibody Fab fragment that binds to an epitope of C1q is identical or overlapping with an epitope of C1q bound by an anti-C1q antibody M1 produced by a hybridoma cell line having ATCC Accession No. PTA-120399. In some embodiments, the humanized anti-C1q antibody Fab fragment competes with another antibody of the present disclosure for binding to C1q. In certain embodiments, the anti-C1q antibody Fab fragment competes with an anti-C1q antibody M1 produced by a hybridoma cell line having ATCC Accession No. PTA-120399 or an anti-C1q binding fragment thereof.

[0039] As is well known in the art, antibody heavy chain and light chain CDR3 domains play a particularly important role in the binding specificity / affinity of antibodies to antigens, and therefore the antibody Fab fragments of the present disclosure may include the heavy chain and light chain CDR3 of the variable region of the monoclonal antibody M1 produced by the hybridoma cell line with ATCC accession number PTA-120399. In some embodiments, the antibody Fab fragment also includes the CDR2 of the variable region of the monoclonal antibody M1. In some embodiments, the antibody Fab fragment also includes the CDR of the variable region of the monoclonal antibody M1. In some embodiments, the antibody Fab fragment may also include any combination of CDRs.

[0040] In some embodiments, the antibody Fab fragment has been engineered to increase brain penetration. In some cases, the antibody Fab fragment has better brain penetration than the corresponding full-length antibody.

[0041] In some embodiments, the antibody Fab fragment has only one binding site. In some embodiments, the antibody Fab fragment has the same affinity for CIq as the full CIq antibody (incorporated herein by reference from U.S. Patent No. 62 / 075793). In some embodiments, the antibody Fab fragment inhibits proteins of the complement cascade, e.g., CIq, C4, C2, C3 convertase, C3a, C5, C3b, C5b, C6, C7, C8, and / or C9.

[0042] In some embodiments, the antibody fragment has a shorter half-life in human circulation compared to the corresponding full-length antibody of the antibody fragment.

[0043] In some embodiments, the antibody Fab fragment is injected directly into the eye. In some embodiments, the antibody Fab fragment is injected into the eye for the prevention or treatment of an eye disease or condition, and can generally be administered by ocular injection, intraocular injection, and / or intravitreal injection. Other methods of administration are also used, including, but not limited to, topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0044] The antibody Fab fragments of the present invention are effective for complement-associated ocular conditions (all ocular conditions and diseases in which complement (including the classical pathway and the alternative pathway) is involved in the pathology) (such as, for example, macular degeneration diseases, such as chronic open-angle glaucoma, acute angle-closure 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 ischemic retinopathy, endophthalmitis and other It is useful for the prevention and treatment of intraocular neovascular diseases such as diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, 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, Pursha retinopathy, Sjogren's dry eye, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, and multiple sclerosis.

[0045] A preferred group of complement-associated ocular 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.

[0046] In some embodiments, the antibody Fab fragment specifically binds to CIq and neutralizes the biological activity of CIq.

[0047] In some embodiments, the antibody Fab fragment has a dissociation constant (K) for human C1q ranging from 10 pM to 20 pM, or 1 pM to less than 10 pM. D In some embodiments, the antibody Fab fragment has a dissociation constant (K) for mouse C1q ranging from 1 pM to 200 pM. D ). In some embodiments, the antibody Fab fragment specifically binds to C1q and inhibits the biological activity of C1q. In some embodiments, the biological activity is (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to phosphatidylserine, (5) C1q binding to pentraxin-3, (6) C1q binding to C-reactive protein (CRP), (7) C1q binding to globular C1q receptor (gC1qR), (8) C1q binding to complement receptor 1 (CR1), (9) C1q binding to β-amyloid protein, (10) C1q binding to calreticulin, (11) C1q binding to apoptotic cells, or (12) C1q binding to components of neural cell membranes.

[0048] In some embodiments, the biological activity is (1) activation of the classical complement activation pathway, (2) activation of antibodies and complement-dependent cytotoxicity, (3) CH50 hemolysis, (4) synaptic loss, (5) B-cell antibody production, (6) dendritic cell maturation, (7) T-cell proliferation, (8) cytokine production, (9) microglial activation, (10) Arthritis reaction, (11) phagocytosis of synapses or nerve terminals, or (12) activation of complement receptor 3 (CR3 / C3) expressing cells.

[0049] In some embodiments, the CH50 hemolysis comprises human CH50 hemolysis, mouse CH50 hemolysis, rat CH50 hemolysis, dog CH50 hemolysis, rhesus monkey CH50 hemolysis, and / or cynomolgus monkey CH50 hemolysis.

[0050] In some embodiments, CH50 hemolysis includes human CH50 hemolysis, mouse CH50 hemolysis and / or rat CH50 hemolysis. In some embodiments, 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 150ng, less than 100ng, less than 50ng, or less than 20ng. In some embodiments, the antibody Fab fragment binds to C1q and inhibits biological function with a binding stoichiometry ranging from less than 6:1 to 1:1 or less than 2:1 to 1:1.

[0051] In some embodiments, the antibody Fab fragment is humanized.

[0052] In one aspect, the disclosure provides host cells that express the Fab fragments of the antibodies of the disclosure.

[0053] In one aspect, the disclosure provides isolated DNA sequences encoding the heavy chain region and / or light chain region of the antibody Fab fragments of the disclosure.

[0054] In one aspect, the disclosure provides cloning or expression vectors comprising one or more DNA sequences of the disclosure.

[0055] In some embodiments, a host cell includes one or more cloning or expression vectors of the disclosure.

[0056] In one aspect, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence encoding an antibody Fab fragment of the present disclosure.

[0057] In one aspect, the present disclosure provides a method for producing the antibody Fab fragment of the present disclosure, the method comprising culturing the host cell of the present disclosure and isolating the antibody Fab fragment.

[0058] In one aspect, the present disclosure provides a pharmaceutical composition comprising the antibody Fab fragment of the present disclosure and a pharmaceutically acceptable carrier.

[0059] In other embodiments, the present disclosure provides an isolated host cell comprising a nucleic acid sequence of any of the preceding embodiments. In some embodiments, the host comprises a cloning or expression vector comprising a nucleic acid sequence of an antibody Fab fragment. In some embodiments, the host cell containing the expression vector and the nucleic acid is cultured under conditions suitable for expression of the antibody Fab fragment. In some embodiments, the antibody Fab fragment is subsequently recovered from the host cell (or host cell culture medium). In addition, a pharmaceutical composition containing an anti-C1q antibody Fab fragment in combination with a pharmaceutically acceptable carrier is provided. The present disclosure also provides a kit containing an anti-C1q antibody Fab fragment for use in any of the methods described herein.

[0060] In one aspect, the present disclosure provides a method of inhibiting synapse loss, the method comprising administering to a patient suffering from adverse synapse loss an antibody Fab fragment selected from the present disclosure.

[0061] In some embodiments, the patient has suffered synaptic loss as a result of a neurodegenerative disease, a central nervous system disorder, or a peripheral nervous system disorder.

[0062] In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0063] In some embodiments, the method further comprises administration of neural progenitor cells or a neurogenesis enhancer.

[0064] In some embodiments, the antibody Fab fragment binds to CIq and inhibits complement activation.

[0065] In one aspect, the disclosure provides methods of treating or preventing a disease associated with complement activation in an individual in need of such treatment, the method comprising administering an antibody Fab fragment of the disclosure.

[0066] In some cases, the disclosure provides methods of treating or preventing a disease associated with complement activation in an individual in need of such treatment, the methods comprising administering an anti-C1q antibody Fab fragment.

[0067] In some embodiments, the disease associated with complement activation is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is associated with the loss of synapses or neural connections (such as synaptic loss dependent on complement receptor 3 (CR3) / C3 or complement receptor CR1). In some embodiments, the neurodegenerative disease is associated with pathological activity-dependent synaptic pruning, synaptic phagocytosis by microglia. In some embodiments, the neurodegenerative disease 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.

[0068] In some embodiments, the neurodegenerative disease is associated with loss of synapses or loss of neural connections.

[0069] In some embodiments, the neurodegenerative disease is associated with synaptic loss that is dependent on complement receptor 3 (CR3) / C3 or complement receptor CR1.

[0070] In some embodiments, the neurodegenerative disease is associated with pathological activity-dependent synaptic pruning.

[0071] In some embodiments, the neurodegenerative disease is associated with synaptic phagocytosis by microglia.

[0072] In some embodiments, the neurodegenerative disease is selected from 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, or Huntington's disease.

[0073] In some embodiments, the disease associated with complement activation is an inflammatory disease, an autoimmune disease, a complement-associated eye disease, or a metabolic disorder.

[0074] In some embodiments, the disease associated with complement activation is an inflammatory disease, autoimmune disease, or metabolic disorder selected from diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), distal tissue damage after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration, age-related macular degeneration (AMD), (AMD-wet), geographic atrophy choroidal neovascularization (CNV), grape Meningitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica (NMO), 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, Pursha retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

[0075] In some embodiments, the disease associated with complement activation is an autoimmune disease selected from myasthenia gravis, type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, paraneoplastic syndromes, vasculitic diseases, hypocomplementemic urticarial vasculitis (HUV), polymyalgia rheumatica, temporal arteritis, Wegener's granulomatosis, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

[0076] In some embodiments, the present disclosure provides a method for detecting synapses in an individual by a) administering an anti-C1q antibody Fab fragment to the individual, and b) detecting the antibody Fab fragment bound to the synapse, thereby detecting the synapse in the individual. In some embodiments, the antibody Fab fragment bound to the synapse is detected using an imaging technique selected from the following: positron emission tomography (PET), X-ray computed tomography, single photon emission computed tomography (SPECT), computed tomography (CT), and computed axial tomography (CAT). In some embodiments, the detection of the antibody Fab fragment bound to the synapse provides a quantitative measurement of the number of synapses in the individual, such that the number of synapses in the individual is repeatedly measured over a period of time, and the loss of synapses in the individual is detected over time, and the loss of synapses over time is a measure of the therapeutic efficacy of a neurodegenerative disease or an autoimmune disease.

[0077] In some embodiments, the present disclosure provides a method for detecting synapses in a biological sample, the method detecting synapses in an individual by a) contacting the biological sample with a humanized anti-C1q antibody Fab fragment, and b) detecting the antibody Fab fragment bound to the synapse. In some embodiments, the method further comprises a step of obtaining a biological sample from the individual prior to step a). In some embodiments, the biological sample comprises a biopsy specimen, tissue, or cell. In some embodiments, the antibody Fab fragment is detected by immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micro-positron emission tomography.

[0078] As used herein, "identity" means that at any particular position in the aligned sequences, the amino acid residues are identical between the sequences. As used herein, "similarity" means that at any particular position in the aligned sequences, the amino acid residues are of a similar type between the sequences. For example, leucine can replace isoleucine or valine. Other amino acids that can be commonly substituted for each other include, but are not limited to:

[0079] - phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

[0080] - lysine, arginine and histidine (amino acids with basic side chains);

[0081] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0082] - Asparagine and glutamine (amino acids with amide side chains); and

[0083] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0084] The degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

[0085] In some embodiments, antibody Fab fragments may be useful in the detection or treatment of a number of diseases or disorders, such as neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barré syndrome (GBS), myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, and Huntington's disease. In some embodiments, antibody Fab fragments may be useful in the detection or treatment of inflammatory diseases, autoimmune diseases, or metabolic disorders, such as diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), distal tissue damage after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, Ig A nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration, age-related macular degeneration (AMD), (AMD-wet), geographic atrophy choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascularization, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica (NMO), 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, Pursha retinopathy, Sjögren's dry eye, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome (COPD), asthma , aspiration pneumonia, myasthenia gravis, type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, paraneoplastic syndromes, vasculitic diseases, hypocomplementemic urticarial vasculitis (HUV), polymyalgia rheumatica, temporal arteritis, Wegener's granulomatosis or multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis).

[0086] In some embodiments, the inflammatory disease, autoimmune disease, complement-associated eye disease, or metabolic disorder is selected from diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), distal tissue damage after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration, age-related macular degeneration (AMD), choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related visual acuity, and retinal dysplasia. Retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica (NMO), 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, Pursha retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

[0087] In one aspect, the disclosure provides a kit comprising an antibody Fab fragment of the disclosure and a package insert comprising instructions for using the antibody Fab fragment to treat or prevent a disease associated with complement activation in an individual in need of such treatment.

[0088] In some embodiments, methods are provided for protecting or treating individuals adversely affected by synaptic loss. These findings have broad implications for a wide variety of clinical conditions, including neurodegenerative conditions involving synaptic loss, where the conditions may include Alzheimer's disease; amyotrophic lateral sclerosis; multiple sclerosis, glaucoma, myotonic dystrophy, Down syndrome; Parkinson's disease, Huntington's disease, etc. The loss of synapses is inhibited by contacting neurons with an agent that blocks complement, including a specific component such as C1q.

[0089] In some embodiments, a method of determining a subject's risk for developing a disease associated with complement activation is provided, the method comprising: (a) administering an anti-C1q antibody fragment to the subject, wherein the antibody fragment is conjugated to a detectable marker; (b) detecting the detectable marker to measure the amount or location of C1q in the subject; and (c) comparing the amount or location of C1q to a reference, wherein based on the comparison of the amount of C1q compared to the reference, the risk of developing a disease associated with complement activation is characterized.

[0090] In some embodiments, detectable markers include nucleic acids, oligonucleotides, enzymes, radioisotopes, biotin or fluorescent markers. In some embodiments, antibodies are labeled with coenzymes (such as biotin) using a biotinylation method. When biotin is used as a marker, detection of the antibody is accomplished by adding a protein (such as avidin or its bacterial counterpart streptavidin, either of which can be combined with a detectable marker (such as dyes, fluorescent markers (such as fluorescein), radioisotopes or enzymes (such as peroxidase) mentioned above).

[0091] In some embodiments, the detectable marker is detected using imaging agents for X-ray, CT, MRI, ultrasound, PET, and SPECT.

[0092] In some embodiments, the fluorescent label is selected from fluorescein, rhodamine, a cyanine dye, or BODIPY.

[0093] In some embodiments, methods are provided for reducing the risk of developing a disease associated with complement activation in a subject, the methods comprising administering an anti-C1q antibody fragment, wherein the anti-C1q 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.

[0094] In some embodiments, the antibody Fab fragment has a shorter half-life compared to the corresponding full-length antibody of the antibody Fab fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 includes two images (A) and (B). Figure 1 shows that M1 Fab has the same binding affinity as M1 whole antibody. Figure 1A ELISA measurement of antibodies binding to CIq (anti-kappa light chain detection) is shown. Figure 1B are images showing M1 whole antibody (bivalent) and M1 Fab (monovalent).

[0096] Figure 2 The M1 Fab was shown to have the same functional potency as the M1 whole antibody. Figure 2 A standard functional measure of inhibition of CIq activity of RBC lysis is shown. DETAILED DESCRIPTION

[0097] Polyclonal and monoclonal antibodies are naturally produced as immunoglobulin (Ig) molecules in the immune system's response to pathogens. The major form, the ~150-kDa IgG1 molecule, with a concentration of 8 mg / ml in human serum, consists of two identical ~50-kDa heavy chains and two identical ~25-kDa light chains.

[0098] Before the advent of recombinant DNA technology, proteolytic enzymes (proteases) that cleave polypeptide sequences have been used to fragment the structure of antibody molecules and determine which parts of the molecule are responsible for their various functions. Restriction digestion with the protease papain cuts the antibody molecule into three fragments. Two fragments (called Fab fragments) are identical and contain antigen binding activity. The Fab fragments correspond to the two identical arms of the antibody molecule, each of which includes the V and B fragments of the heavy chain. H and C H The Fab fragments are characterized by their high mobility and tissue penetration, their reduced circulation half-life, their monovalent ability to bind antigen without mediating antibody effector functions, and their low immunogenicity, which are superior to IgG for certain in vivo applications.

[0099] Fab molecules have constant domains C H 2 and C H 3 Artificial ~50-kDa fragment of Ig molecule with shortened heavy chain. Two heterophilic (V L -V H and C L -C H 1) Domain interaction forms the basis of the two-chain structure of the Fab molecule, which is connected by C L and C H Fab and IgG have the same antigen binding site, which is composed of six complementary determining regions (CDRs) (from V L and V H The CDRs define the highly variable antigen binding site of the antibody. The highest sequence variation is found in LCDR3 and HCDR3. In the natural immune system, LCDR3 and HCDR3 are expressed by V L and J L Gene or V H , D H and J HThe rearrangement of genes generates. LCDR3 and HCDR3 usually form the core of the antigen binding site. The conserved region that connects and displays six CDRs is called the framework region. In the three-dimensional structure of the variable domain, the framework region forms a sandwich of two relative antiparallel β-folds connected by the hypervariable CDR loops on the outside and by the conservative disulfide bridges on the inside. The stability of the antigen binding site of Fab and IgG and this unique combination of versatility constitutes the basis for its success in clinical practice for diagnosis, monitoring, prevention and treatment of diseases.

[0100] In certain embodiments, the present disclosure provides an anti-C1q antibody Fab fragment comprising a heavy chain (V H / C H 1) and light chain (V L / C L ) of the C1q protein, wherein the anti-C1q antibody Fab fragment has six complementary determining regions (CDRs) from V L and V H Three each (HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3). The heavy chain of the antibody Fab fragment is truncated after the first heavy chain domain of IgG1 (SEQ ID NO: 1) and includes the following amino acid sequence:

[0101]

[0102] The complementarity determining regions (CDRs) of SEQ ID NO: 1 are depicted in bold and underlined text.

[0103] The nucleotide sequence corresponding to SEQ ID NO: 1 is SEQ ID NO: 3:

[0104]

[0105] The complementarity determining regions (CDRs) of SEQ ID NO: 3 are depicted in bold and underlined text.

[0106] The light chain domain of the antibody Fab fragment includes the following amino acid sequence (SEQ ID NO: 2):

[0107]

[0108] The complementarity determining regions (CDRs) of SEQ ID NO: 2 are depicted in bold and underlined text.

[0109] The nucleotide sequence corresponding to SEQ ID NO:2 is SEQ ID NO:4:

[0110]

[0111] The complementarity determining regions (CDRs) of SEQ ID NO: 4 are depicted in bold and underlined text.

[0112] The amino acid sequence corresponding to SEQ ID NO:5 is:

[0113] The complementarity determining regions (CDRs) of SEQ ID NO: 5 are depicted in bold and underlined text.

[0114] The amino acid sequence corresponding to SEQ ID NO:6 is:

[0115] The complementarity determining regions (CDRs) of SEQ ID NO: 6 are depicted in bold and underlined text.

[0116] The amino acid sequence corresponding to SEQ ID NO:7 is:

[0117] The complementarity determining regions (CDRs) of SEQ ID NO: 7 are depicted in bold and underlined text.

[0118] The amino acid sequence corresponding to SEQ ID NO:8 is:

[0119] The complementarity determining regions (CDRs) of SEQ ID NO: 8 are depicted in bold and underlined text.

[0120] The amino acid sequence corresponding to SEQ ID NO:9 is:

[0121] The complementarity determining regions (CDRs) of SEQ ID NO:9 are depicted in bold and underlined text.

[0122] The amino acid sequence corresponding to SEQ ID NO: 10 is:

[0123] The complementarity determining regions (CDRs) of SEQ ID NO: 10 are depicted in bold and underlined text.

[0124] The amino acid sequence corresponding to SEQ ID NO: 11 is:

[0125] The complementarity determining regions (CDRs) of SEQ ID NO: 11 are depicted in bold and underlined text.

[0126] The amino acid sequence corresponding to SEQ ID NO: 12 is:

[0127] The complementarity determining regions (CDRs) of SEQ ID NO: 12 are depicted in bold and underlined text.

[0128] Methods for protecting or treating individuals who suffer from the adverse effects of synaptic loss are provided. It is demonstrated herein that normally developing immature astrocytes produce signals that induce neurons to express specific complement proteins, thereby opening a developmental window during which synaptic clearance occurs. The expression of these proteins during development reflects the period of developmental synaptogenesis, which is off in the embryonic and adult brains, but at high levels in the postnatal brain.

[0129] These findings have broad implications for a wide variety of clinical conditions, particularly neurodegenerative conditions in which synapse loss is involved. Synapse loss is inhibited by contacting neurons with inhibitors or antagonists of the complement pathway. For example, inhibitors can block activation of the complement cascade, can block 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 the adult brain) is of great significance for maintaining normal neuronal function in a wide variety of neurodegenerative conditions.

[0130] definition

[0131] As used in the specification, "a" or "an" may mean one or more. As used in one or more claims, when used with the word "comprising", the word "a" or "an" may mean one or more than one. For example, a reference to an "antibody" is a reference to one or more antibodies, and includes equivalents thereof known to those skilled in the art. As used herein, "another" may mean at least a second or more.

[0132] The term "prevention" is art-recognized and well understood in the art when used in connection with a condition such as epilepsy, and includes the administration of a composition that reduces the frequency or severity of one or more symptoms of a medical condition in a subject, or delays the onset of one or more symptoms of a medical condition in a subject, relative to a subject not receiving the composition. Thus, prevention of the progression of epilepsy includes, for example, slowing or stopping the average amount of neurodegeneration in a treated patient population, for example, by a statistically and / or clinically significant amount, relative to a control population not receiving treatment. Similarly, prevention of the progression of a neurodegenerative disease includes reducing the likelihood that a treated patient will develop an injury (such as cognitive impairment and / or memory loss), or delaying the onset of an injury, relative to an untreated patient.

[0133] As used herein, the term "subject" refers to a living mammal and can be used interchangeably with the term "patient". Examples of mammals include, but are not limited to, any member of the class mammals: humans, non-human primates (such as chimpanzees and other apes and monkeys); livestock (such as cattle, horses, sheep, goats, pigs); domestic animals (such as rabbits, dogs and cats); laboratory animals (including rodents, such as rats, mice and guinea pigs), etc. The term does not indicate a particular age or sex.

[0134] As used herein, the terms "treating" or "treatment" encompass reducing, arresting or reversing the symptoms, clinical signs or underlying pathology of a condition to stabilize or improve the subject's condition, or to reduce the likelihood that the subject's condition will worsen as if the subject were not receiving treatment.

[0135] For the subject treatment methods, the term "therapeutically effective amount" of a compound refers to an amount of one or more compounds in a formulation that, when the formulation is administered (to a mammal (preferably a human) as part of a desired dosing regimen, alleviates symptoms, lessens the condition, or slows the onset of a disease condition, according to clinically acceptable standards for the disorder or condition to be treated or cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutically effective amount herein may vary according to factors such as the patient's disease condition, age, sex, and weight, and the ability of the antibody to elicit a desired response in the individual.

[0136] As used herein, an individual who is "at risk" of developing a particular disease, disorder, or condition may or may not have detectable symptoms of the disease or disease, and may or may not have exhibited detectable symptoms of the disease or disease, prior to the treatment methods described herein. As known in the art, "at risk" indicates that an individual has one or more risk factors, which are measurable parameters associated with the development of a particular disease, disorder, or condition. An individual who has one or more of these risk factors has a higher likelihood of developing a particular disease, disorder, or condition than an individual who does not have one or more of these risk factors.

[0137] "Chronic" administration refers to administration of one or more drugs in a continuous rather than acute mode to maintain the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration refers to treatment that is not continuous administration without breaks, but rather cyclical / periodic in nature.

[0138] As used herein, administration "in conjunction" with another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also includes administration as a co-formulation or administration as a separate composition, including with different dosing frequencies or intervals, and using the same route of administration or different routes of administration.

[0139] Synapse loss. Synapses are asymmetric communication connections formed at the neuromuscular junction (NMJ) between two neurons or between neurons and muscle cells. Chemical synapses achieve cell-to-cell communication via the secretion of neurotransmitters, while in electrical synapses, signals are transmitted through gap junctions (specialized intercellular channels that allow ionic currents to flow). In addition to ions, other molecules that regulate synaptic function (such as ATP and second messenger molecules) can diffuse through gap junction holes. At mature NMJs, the presynaptic and postsynaptic membranes are separated by the synaptic cleft containing extracellular proteins that form the basement membrane. Synaptic vesicles are aggregated at presynaptic release sites, transmitter receptors are aggregated in the junctional folds at the postsynaptic membrane, and glial processes surround the nerve endings.

[0140] Synaptogenesis is a dynamic process. During development, more synapses are established than the synapses that will eventually be retained. Therefore, the removal of excessive synaptic input is a key step in the maturation of synaptic circuits. Synaptic clearance is a competitive process involving the interaction between presynaptic partners and postsynaptic partners. In the CNS, as with the NMJ, the activity-dependent reconstruction of the development of synaptic circuits occurs through the process of selective stabilization of coactive inputs and the removal of inputs with irrelevant activity. Through the dynamic process involving the rapid removal of synapses, the anatomical refinement of synaptic circuits occurs at the level of individual axons and dendrites. Along with axonal branching and reconstruction, synapses are formed and stripped using the rapid synaptic removal that occurs.

[0141] In addition to normal developmental loss, synaptic loss is a common early pathological event in many neurodegenerative diseases and is most relevant to cognitive impairment. In the brains of patients with preclinical Alzheimer's disease (AD), and studies in transgenic animal models have demonstrated that synaptic damage occurs early in the progression of the disease. Early destruction of this synaptic connection in the brain causes neuronal dysfunction, which in turn leads to the typical symptoms of dementia and / or movement disorders observed in several neurodegenerative diseases.

[0142] Several molecules involved in AD and other neurodegenerative diseases play an important role in synaptic function. For example, AβPP has a preferential localization in central and peripheral synaptic sites. In transgenic mice, abnormal expression of mutant forms of AβPP not only causes amyloid deposition, but also causes extensive synaptic damage. This synaptic pathology occurs at an early stage and is related to the level of soluble Aβ1-42 rather than plaque formation. Other neurodegenerative diseases in which gene products have been shown to be closely related to 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 the synaptic vesicle protein synaptophysin. Studies in the human brain have detected htt in the perikarya, neuropil, varicosity, and possibly nerve terminals of some neurons based on punctate staining. Serine / threonine kinase (DMK) is the gene product of the DM gene, which has been found to be located postsynaptic at the neuromuscular junction of skeletal muscle and postsynaptic at the intercalated disc of cardiac tissue. DMK is also found at synaptic sites in the cerebellum, hippocampus, midbrain, and medulla.

[0143] Inhibition of synaptic loss results in the maintenance of synapses or a reduced loss, but the reduction will occur in another way. As used herein, by "regulation" of synaptic loss, it is meant that the number of lost synapses is enhanced or inhibited, as required in the specific situation. As used herein, the term "regulator of synaptic loss" refers to an agent that is capable of altering synaptic loss. Regulators include, but are not limited to, both "activators" and "inhibitors." "Activators" or "agonists" are substances that enhance synaptic loss. Conversely, "inhibitors" or "antagonists" reduce synaptic loss. The reduction can be complete or partial. As used herein, regulators include (but are not limited to) C1q antagonists and agonists.

[0144] Agonists and antagonists can comprise proteins, nucleic acids, carbohydrates, antibodies or any other molecules that reduce the effect of proteins. The term "analog" is used herein to refer to a molecule that is structurally similar to a molecule of interest but has been modified in a targeted and controlled manner by replacing a specific substituent of a reference molecule with a substituent for replacement. Analogs can exhibit identical, similar or improved effects compared to the starting molecule. Synthesis and screening of analogs to identify variants of known compounds with improved properties (such as higher efficacy to specific receptor types, or higher selectivity to targeted receptor types and lower activity levels to other receptor types) is a well-known method in medicinal chemistry.

[0145] Complement. Complement is a system of plasma proteins that interact with the cell surface of pathogens or cells to mark them for destruction by phagocytes. The complement system consists of a large number of different plasma proteins produced primarily by the liver. Some of these proteins are a class of proteases, called zymogens, which themselves are activated by proteolytic cleavage. Therefore, these zymogens can be widely distributed and not active until activated by local pathogens. The complement system is therefore activated by a triggered enzyme cascade.

[0146] The classical pathway is activated by direct binding of the complement protein C1q to the cell surface or to an antibody bound to the cell surface. C1q is a large multimeric protein of 460 kDa, consisting of 18 polypeptide chains (6 C1q A chains, 6 C1q B chains and 6 C1q C chains). C1r and C1s complement proteins bind to the C1q tail region to form a C1 complex. The binding of the C1q complex to the surface of the cell or to the complement binding domain of the Fc region of the antibody induces a conformational change in C1q, which leads to the activation of the autocatalytic enzyme activity in C1r, which then cuts C1s to generate an active serine protease. Once activated, C1s cuts C4, etc., leading to a complement cascade sequence. Ultimately, this pathway leads to the formation of a membrane attack complex that lyses and kills infected cells. Normal cells (including neurons) express molecules such as CD59 (which protects normal cells from lysis or damage from the membrane attack complex) and C1 inhibitors (CI-INH) (which dissociate C1r and C1s from the active C1 complex).

[0147] Various complement proteins are expressed in vitro and in vivo by neurons and glial cells. Their functions in the brain are 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 C1q, C1r, C1s, C4, C2 and C3, as well as more terminal proteins. Neurons have been reported to express C4 and C3, but only C1q after brain injury.

[0148] It has been clarified that the complement cascade can be triggered by three pathways: the classical pathway, the alternative pathway, and the lectin pathway. All three pathways converge through a common intersection (complement C3). C3 is an acute phase reactant. Although a small amount is also produced by activated monocytes and macrophages, the liver is the main site of synthesis. A single-chain precursor (pro-C3) of approximately 200 kD is found in the cell; eDNA shows that it includes 1663 amino acids. This is processed into α subunits and β subunits by proteolytic cleavage, and in the mature protein, the α subunit and the β subunit are connected by a disulfide bond. Pro-C3 contains a signal peptide of 22 amino acid residues, a β chain (645 residues) and an α chain (992 residues). The two chains are connected by 4 arginine residues (not present in the mature protein). In the alternative pathway, complement C3 undergoes spontaneous cleavage, causing complement B to bind to C3b. The diffusion of the Ba subunit causes the active alternative pathway C3 convertase (C3bBb). C3bBb is stabilized by binding to properdin prior to confluence.

[0149] Inhibition of complement. Many molecules are known to inhibit the activity of complement. In addition to known compounds, suitable inhibitors can be screened by the methods described herein. As described above, normal cells can produce proteins that block complement activity, for example, CD59, C1 inhibitor, etc. In some embodiments of the invention, complement is inhibited by upregulating the expression of genes encoding such polypeptides.

[0150] Modification of molecules that block complement activation is also known in the art. Such molecules include (but are not limited to) modified complement receptors, such as soluble CR1. The most common allotype of mature protein of CR1 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 consists of 30 repeating units called short consensus repeats (SCR) or complement control protein repeats (CCPR), each of which consists of 60 to 70 amino acid residues. Recent data show that C1q specifically binds to human CR1. Therefore, CR1 recognizes all three complement opsonins, namely C3b, C4b and C1q. A soluble form of recombinant human CR1 (sCR1) lacking the transmembrane domain and the cytoplasmic domain has been produced and has been shown to retain all known functions of natural CR1. The cardioprotective effect of sCR1 has been confirmed in animal models of ischemia / reperfusion injury. Several types of human C1q receptors (C1qR) have been described. These include a ubiquitous 60-67 kDa receptor, referred to as cC1qR because it binds to the collagen-like domain of C1q. This C1qR variant was shown to be calreticulin; a 126 kDa receptor that regulates monocyte phagocytosis. gC1qR is not a membrane-bound molecule, but a secreted soluble protein with affinity for the globular region of C1q and may serve as a fluid-phase regulator of complement activation.

[0151] Decay accelerating factor (OAF) (CD55) consists of four SCRs plus a serine / threonine-rich domain capable of extensive O-linked glycosylation. OAF is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, and through the ability of OAF to bind C4b and C3b, it acts by dissociating C3 and C5 convertases. The soluble form of OAF (sDAF) has been shown to inhibit complement activation.

[0152] C1 inhibitor, a member of the "serpin" family of serine protease inhibitors, is a heavily glycosylated plasma protein that prevents fluid phase C1 activation. C1 inhibitor regulates the classical pathway of complement activation by blocking the active sites of C1r and C1s and dissociating them from C1q.

[0153] Peptide inhibitors of complement activation include C5a (van Oostrum et al., 1996); C5a C-terminal octapeptide (Kawai et al., 1992); C5a His67 modified C-terminal octapeptide analog (Or et al., 1992); C089 (C5a hexapeptide, Konteatis et al., 1994); C3a C-terminus (Kretzschmar et al., 1992); factor B-related hexapeptide (Lesavre et al., 1982); C1q B chain helical region (Fryer et al., 1997); DFP (diisopropyl fluorophosphate, 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); FUT-175 (nafamostat mesylate, Inose et al., 1997).

[0154] Other inhibitory molecules include deoxygalactan (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).

[0155] The term "complement-associated ocular conditions" is used in the broadest sense and encompasses all ocular conditions in which complement is involved in the pathology, including the classical and alternative pathways of complement, and in particular the alternative pathway. Complement-associated ocular conditions include, but are not limited to, chronic open-angle glaucoma, acute angle-closure 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 retinopathy, and other intraocular neovascular diseases such as diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, 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, Pursha retinopathy, Sjogren's dry eye, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, and multiple sclerosis.

[0156] A preferred group of complement-associated ocular 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.

[0157] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The term "antibody" herein is used in the broadest sense, and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments (as long as they exhibit the desired biological activity).

[0158] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. H and V L Paired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0159] The L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes or isotypes based on the amino acid sequence of the constant domain of their heavy chains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha ("α"), delta ("δ"), epsilon ("ε"), gamma ("γ"), and mu ("μ"), respectively. Based on relatively minor differences in CH sequence and function, the gamma and alpha classes are further divided into subclasses (isotypes), for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and described, for example, in Abbas et al., Cellular and Molecular Immunology, 4 th ed. (WB Saunders Co., 2000).

[0160] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds in the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V H ), followed by multiple constant domains. Each light chain has a variable domain at one end (V L ), with 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. Specific amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0161] An "isolated" molecule or cell is one that has been identified and separated from at least one contaminating molecule or cell with which it is normally associated in the environment in which it is produced. Preferably, the isolated molecule or cell is not associated with all components associated with the production environment. The isolated molecule or cell is in a form that is different from the form or state in which it is found in nature. Thus, an isolated molecule is different from a molecule that naturally occurs in a cell; an isolated cell is different from a cell that naturally occurs in a tissue, organ, or individual. In some embodiments, the isolated molecule is an anti-C1q antibody Fab fragment of the present disclosure. In other embodiments, the isolated cell is a host cell or hybridoma cell that produces an anti-C1q antibody Fab fragment of the present disclosure.

[0162] An "isolated" antibody is one that has been identified, separated, and / or recovered (e.g., native or recombinant) from a component of its production environment. Preferably, an isolated polypeptide is free of association with all other contaminating components from its production environment. Contaminating components from its production environment, such as those resulting from recombinant transfected cells, are materials that will generally interfere with research, diagnostic, or therapeutic applications of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of the antibody (as determined by, e.g., 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 Brilliant Blue, or preferably silver stain). An isolated antibody comprises the antibody in situ within a recombinant T-cell, since at least one component of the antibody's natural environment will not be present. However, isolated polypeptide or antibody will ordinarily be prepared by a method that includes at least one purification step.

[0163] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L These domains are usually the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding site.

[0164] The term "variable" refers to the fact that certain segments of the variable domain between antibodies are widely different in sequence. The V domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed over the entire span of the variable domain. On the contrary, in both the light chain variable domain and the heavy chain variable domain, variability is concentrated in three segments referred to as hypervariable regions (HVRs). The more highly conserved parts of the variable domain are referred to as framework regions (FRs). The variable domains of native heavy and light chains each include four FR regions, which mainly adopt a β-folded configuration, are connected by forming three HVRs connected in a loop, and form a part of the β-folded structure in some cases. The HVRs in each chain are kept close to each other by the FR region, and together with the HVRs from other chains, promote the formation of an antibody's antigen binding site (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domain does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions, such as the participation of antibodies in antibody-dependent cellular toxicity.

[0165] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen binding sites found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. 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. 262: 732-745 (1996), where definitions include overlaps and subsets of amino acid residues when compared to each other. However, the application of any definition of CDR referring to an antibody or a transplanted antibody or its variant is intended to be within the scope of the term defined and used herein. The amino acid residues comprising each defined CDR in the references cited above are listed in Table (X) below as a control. The CDR listed in Table (X) is defined according to Kabat 1991.

[0166] As used herein, the terms "CDR-L1", "CDR-L2" and "CDR-L3" refer to the first CDR, the second CDR and the third CDR in the light chain variable region, respectively. As used herein, the terms "CDR-H1", "CDR-H2" and "CDR-H3" refer to the first CDR, the second CDR and the third CDR in the heavy chain variable region, respectively. As used herein, the terms "CDR-1", "CDR-2" and "CDR-3" refer to the first CDR, the second CDR and the third CDR of the variable region of either chain, respectively.

[0167] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies of the population are identical, except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Different from polyclonal antibody preparations that typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" means that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and is not interpreted as requiring the production of antibodies by any particular method. For example, the monoclonal antibodies to be used according to the present invention can 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, 2ded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., Nature, 353:625-628 (1992); al., J.Mol.Biol.222:581-597(1992); Sidhu et al., J.Mol.Biol.338(2):299-310(2004); Leeet al., J.Mol.Biol.340(5):1073-1093(2004); Fellouse, Proc.Nat'l Acad. Sci. USA 101(34):12467-472(2004); and Lee et al., J. Immunol.Methods 284(1-2):119-132 (2004)) and techniques for producing human or human-like antibodies in animals having human immunoglobulin loci or genes encoding part or all of human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Nat'l Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). .

[0168] The terms "full length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as distinguished from an antibody fragment. In particular, whole antibodies include those having heavy and light chains that include 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. In some cases, intact antibodies may have one or more effector functions.

[0169] "Antibody fragments" include a portion of an intact antibody, preferably, the antigen binding region and / or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 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.

[0170] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a name that reflects the ability to crystallize readily. The Fab fragment consists of the entire L chain and the variable region (V H ), and the first constant domain of a heavy chain (C H 1) Together. With respect to antigen binding, each Fab fragment is monovalent, i.e., it has a single antigen binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen binding activities and is still able to cross-link antigen. The Fab' fragment differs from the Fab fragment in that C H The constant domains have a small number of additional residues at the carboxyl terminus, 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 carry free sulfhydryl groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that had hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0171] The Fc fragment includes the carboxyl terminal portions of two H chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0172] The term "Fc region" herein is used to define the C-terminal region (comprising native sequence Fc region and variant Fc region) of an immunoglobulin heavy chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as the amino acid residue at position Cys226, or extends to its carboxyl terminus from Pro230. The C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of an antibody, or by recombinant engineering encoding the nucleic acid of the heavy chain of an antibody. Accordingly, the composition of a complete antibody can include an antibody population that removes all K447 residues, an antibody population that does not remove the K447 residue, and an antibody population with a mixture of K447 residues and antibodies that do not have the K447 residue. Suitable native sequence Fc regions for use in antibodies of the present invention include human IgG1, IgG2, IgG3, and IgG4.

[0173] A "native sequence Fc region" includes an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0174] A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by virtue of at least one amino acid modification (preferably one or more amino acid substitutions). Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, for example from about one to about ten amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide, and preferably from about one to about five amino acid substitutions. The variant Fc region herein will preferably have at least about 80% homology with the native sequence Fc region and / or with the Fc region of the parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0175] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is a receptor that binds to an IgG antibody (gamma receptor), and comprises receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternative splicing forms of these receptors), the FcγRII receptor comprising FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), FcγRIIA and FcγRIIB having similar amino acid sequences (mainly differing in their cytoplasmic domains). The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in its cytoplasmic domain. (See, e.g., M. 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 may also increase the serum half-life of antibodies.

[0176] Human FcRn high affinity binding polypeptides can be assayed for binding to FcRn in vivo and serum half-life, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with variant Fc regions. WO 2004 / 42072 (Presta) describes antibody variants with enhanced or reduced binding to FcR. See also, for example, Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001).

[0177] "Fv" is the smallest antibody fragment containing a complete antigen recognition and antigen binding site. The fragment consists of a dimer of a heavy chain variable region and a light chain variable region in a tight, non-covalent association. Six hypervariable loops (3 loops from the H chain and 3 loops from the L chain) emanate from the folding of these two domains, which provide amino acid residues for antigen binding and give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv, which includes only three HVRs specific for an antigen) has the ability to recognize and bind to an antigen (although with lower affinity than the entire binding site).

[0178] "Single-chain Fv", also referred to as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains linked to a single polypeptide chain. Preferably, the sFv polypeptide also comprises V H Domain and V L A polypeptide linker between domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0179] The "functional fragment" of an antibody includes a portion of an intact antibody, which generally includes the antigen binding region or variable region of the intact antibody, or the F region of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0180] The term "diabodies" refers to small antibody fragments that are prepared by using V H Domain and V LsFv fragments (see the preceding paragraph) are constructed by using short linkers (about 5-10 residues) between the V domains to achieve interchain rather than intrachain pairing of the V domains, thereby generating bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossed" sFv fragments, in which the V domains of the two antibodies are H Domain and V L The domains are present on different polypeptide chains. Dimers are described in more detail in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48 (1993).

[0181] As used herein, "chimeric antibodies" refer to antibodies (immunoglobulins) in which a portion of the heavy and / or light chains is identical 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 one or more chains is identical 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, as long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81: 6851-55 (1984)). The chimeric antibodies of interest herein include Antibodies, wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing a macaque with an antigen of interest. As used herein, "humanized antibodies" are a subset of "chimeric antibodies."

[0182] The "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (acceptor antibody), wherein the residues from the HVR of the acceptor are replaced by residues from the HVR (donor antibody) of a non-human species (e.g., mouse, rat, rabbit, or non-human primate) with desired specificity, affinity, and / or capacity. In some cases, the FR residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies can include residues not found in the acceptor antibody or in the donor antibody. These modifications can be performed to further improve antibody performance, such as binding affinity. Typically, a humanized antibody will include substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences, although the FR regions may contain one or more single FR residue substitutions that improve antibody performance (such as binding affinity, isomerization, immunogenicity, etc.). The number of these amino acid substitutions in the FR is usually no more than 6 on the H chain and no more than 3 on the L chain. The humanized antibody will also optionally include at least a portion of an immunoglobulin constant region (Fc) (usually a constant region of a human immunoglobulin). For more details, see, for example, Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994) and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0183] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or that has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that include 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). Methods that can be used to prepare human monoclonal antibodies are also described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been engineered to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disrupted, e.g., an immune xenomice (see, e.g., XENOMOUSE TM Nos. 6,075,181 and 6,150,584 for the production of human antibodies via human B cell hybridoma technology). See also, e.g., Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006) for the production of human antibodies via human B cell hybridoma technology.

[0184] When used in this article, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain that is hypervariable and / or forms a structurally determined loop in a sequence. Typically, an antibody includes six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the greatest difference of the six HVRs, and H3 is particularly considered to play a unique role in conferring precise specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0185] There are a variety of HVR descriptions in use and included herein. HVRs that are Kabat complementary determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., above). In contrast, Chothia refers to the position of the structural loop (Chothia and Lesk J.Mol.Biol.196:901-917 (1987)). AbM HVRs embody a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. The residues from each of these HVRs are as follows.

[0186]

[0187] HVRs may include "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (preferred embodiments) (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these extended HVR definitions, the variable domain residues are numbered according to Kabat et al., supra.

[0188] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0189] The phrase "Kabat numbering of variable domain residues" or "Kabat numbering of amino acid positions" and variations thereof refer to the numbering system used for heavy chain variable domains or light chain variable domains compiled in Kabat et al. above. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion of the FR or HVR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). For a given antibody, the Kabat numbering of the residues can be determined by aligning the homologous region of the antibody sequence with the sequence of the "standard" Kabat numbering.

[0190] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported in Kabat et al. above). The "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, reference to the residue numbering in the variable domain of an antibody means the residue numbering according to the Kabat numbering system. Unless otherwise specified herein, reference to the residue numbering in the constant domain of an antibody means the residue numbering according to the EU numbering system (e.g., see, U.S. Patent Publication No. 2010-280227).

[0191] As used herein, "acceptor human framework" is a framework comprising an amino acid sequence of a VL framework or a VH framework derived from a human immunoglobulin framework or a human common framework. The acceptor human framework "derived from" a human immunoglobulin framework or a human common framework may include an amino acid sequence identical thereto, or it may contain a pre-existing amino acid sequence change. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In the case where pre-existing amino acid changes are present in VH, preferably those changes occur only at three, two or one of positions 71H, 73H and 78H; for example, the amino acid residues at those positions may be 71A, 73T and / or 78A. In one embodiment, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or a human common framework sequence.

[0192] A "human common framework" is a framework that represents the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH framework sequences is from a subgroup of variable domain sequences. Typically, a subgroup of sequences is a subgroup as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples thereof include, for VL, a subgroup may be subgroup kappa I, kappa II, kappa III, or kappa IV as described above in Kabat et al. In addition, for VH, a subgroup may be subgroup I, subgroup II, or subgroup III as described above in Kabat et al.

[0193] "Amino acid modification" at a specific position refers to the replacement or deletion of a specific residue, or the insertion of at least one amino acid residue near a specific residue. Insertion "near" a specific residue means insertion within one to two residues thereof. Insertion can be at the N-terminus or C-terminus of a specific residue. Preferred amino acid modifications herein are replacements.

[0194] An "affinity matured" antibody is one that has one or more alterations in one or more of its HVRs that result in an improved affinity of the antibody for the antigen compared to a parent antibody that does not have those one or more alterations. In one embodiment, 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 shuffling of VH and VL domains. Random mutagenesis of HVRs and / or framework residues is described, for example, by Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995) and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0195] As used herein, the term "specific recognition" or "specific binding" refers to a measurable and reproducible interaction (such as attraction or binding between a target and an antibody) that is decisive for determining the presence of a target in the presence of a heterogeneous population of molecules comprising biological molecules. For example, an antibody that specifically or preferentially binds to a target or epitope is an antibody that binds to the target or epitope with greater affinity, avidity, more readily and / or for a longer duration than the antibody binds to other targets or other epitopes of the target. It is also understood that, for example, an antibody (or portion) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Likewise, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. An antibody that specifically binds to a target may have 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 embodiments about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 Up to 10 9 M-1 , or about 10 10 M -1 Up to 10 11 M -1 or higher association constant. Various immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immunoreact with proteins. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0196] As used herein, an "interaction" between a complement protein and a second protein includes, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, binding, covalent binding, and ionic binding. As used herein, an antibody "inhibits" an "interaction" between two proteins when the antibody disrupts, reduces, or completely eliminates the interaction between the two proteins. An antibody or fragment thereof of the present disclosure "inhibits" an "interaction" between two proteins when the antibody or fragment thereof binds to one of the two proteins.

[0197] A "blocking" antibody, "antagonist" antibody, "inhibitory" antibody, or "neutralizing" antibody is an antibody that inhibits or reduces one or more biological activities (such as interaction with one or more proteins) of an antigen to which it binds. In some embodiments, a blocking antibody, antagonist antibody, inhibitory antibody, or "neutralizing" antibody substantially or completely inhibits one or more biological activities or interactions of an antigen.

[0198] 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.

[0199] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., antibody and antigen), and is expressed as a dissociation constant (KD). Affinity can be at least 1 times greater than the affinity of the antibody for an unrelated amino acid sequence, at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, at least 100 times greater, or at least 1000 times greater, or greater. The 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. As used herein, the term "antibody avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. For antibodies and / or antigen binding fragments, the terms "immunoreactivity" and "preferential binding" are used interchangeably herein.

[0200] The term "binding" refers to a direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). The subject anti-C1s antibodies specifically bind to an epitope within the complement C1s protein. "Specific binding" refers to a binding affinity of at least about 10 -7 M or larger (e.g., 5×10 -7 M, 10 -8 M, 5×10 -8 "Non-specific binding" refers to binding with an affinity of less than about 10 -7 M binding affinity, for example, has 10 - 6 M, 10 -5 M, 10 -4 M and other affinity binding.

[0201] As used herein, the term “k on ” refers to the rate constant for the association of an antibody with an antigen.

[0202] As used herein, the term “k off ” refers to the rate constant for the dissociation of an antibody from an antibody / antigen complex.

[0203] As used herein, the term "K D ” refers to the equilibrium dissociation constant of the antibody-antigen interaction.

[0204] As used herein, "percentage (%) of amino acid sequence identity" and "homology" for peptide, polypeptide or antibody sequences refer to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (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.

[0205] "Biological sample" includes a variety of sample types obtained from an individual, and can be used for diagnosis or monitoring determination. The definition includes blood and other liquid samples of biological origin, solid tissue samples (such as biopsy specimens or tissue cultures or cells derived therefrom) and their progeny. The definition also includes samples that have been manipulated in any way after acquisition, such as by treatment with reagents, dissolution or enrichment of certain components (such as polynucleotides). The term "biological sample" includes clinical samples, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, intraocular fluid, synovial fluid, blood components (such as plasma and serum), etc. The term "biological sample" also includes solid tissue samples, tissue culture samples and cell samples.

[0206] An "isolated" nucleic acid molecule is one that has been identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, the isolated nucleic acid is not associated with all components associated with the environment in which it is produced. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in a form that is different from the form or state in which they are found in nature. Thus, the isolated nucleic acid molecules are different from the nucleic acids encoding the polypeptides and antibodies herein that are naturally present in cells.

[0207] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid that has been connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA, to which additional DNA segments can be connected. Another type of vector is a bacteriophage vector. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors", or simply as "expression vectors". Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably as plasmid is the most common form of vector.

[0208] As used interchangeably in the text, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or can be incorporated into any substrate in a polymer by DNA or RNA polymerase or by synthesis reactions. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure (if present) can be imparted before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can include one or more modifications performed after synthesis, such as being conjugated to a marker. Other types of 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 bonds (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), those containing alkylating agents, those with modified bonds (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of one or more polynucleotides. In addition, any hydroxyl group that is usually present in sugars can be, for example, phosphonate group, phosphate group replacement, protected by standard protecting groups, or activated to prepare additional keys with additional nucleotides, or can be conjugated to solid or semisolid supports. 5' and 3' end OH can be phosphorylated or can be partially replaced by amine or organic capping groups from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose), acyclic analogs, and alkaline nucleoside analogs (such as methyl riboside). One or more phosphodiester bonds can be replaced by alternative linking groups.These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. All bonds in a polynucleotide need not be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0209] "Host cell" includes individual cells or cell cultures that can be or have been recipients of one or more vectors for integration of polynucleotide insertions. 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 mutations. Host cells include cells transfected in vivo with one or more polynucleotides of the invention.

[0210] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer that is non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Typically, a physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers (such as phosphates, citrates, and other organic acids); antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, 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 TWEEN TM , polyethylene glycol (PEG) and PLURONICS TM ).

[0211] As used herein, the term "about" refers to the usual error range for each value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments for that value or parameter per se.

[0212] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods or materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials associated with the cited publications. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3 edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMAusubel, et al. eds., (2003)); theseries Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GRTaylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJGait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(JECellis,ed.,1998)Academic Press; Animal Cell Culture (RIFreshney), ed., 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-8) J.The widely used methods described in Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988 - 1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

[0213] Therapeutic methods

[0214] The methods of the present invention provide for the regulation of immune responses to the diseases disclosed herein by administering an agent that is an antagonist of complement. For example, without being bound by theory, during development, immature astrocytes induce the expression of C1q protein in neurons. Inflammatory mediators (such as complement factors) are normally expressed at very low levels in healthy brain tissue, but can be rapidly induced by a variety of attacks on the brain (such as infection, local ischemia and injury). Activation of C1q, C1r and C1s contributes to the inflammatory response, which leads to synaptic loss, accompanied by the generation and recurrence of epileptic seizures and neuronal damage associated with epileptic seizures. During the development of neurodegenerative diseases, overexpression of C1q, C1r and C1s can be coupled with signals of complement activation (e.g., β-amyloid, APP, cytokines (such as IFNγ, TNFα), etc.) that also cause inflammation.

[0215] By administering agents that inhibit complement activation, synapses that would otherwise be lost can be maintained. Such agents include anti-C1q antibody Fab fragment inhibitors, agents that upregulate the expression of natural complement inhibitors, agents that downregulate the synthesis of C1q, C1r or C1s in neurons, agents that block complement activation, agents that block complement activation signals, and the like.

[0216] The method promotes improved maintenance of neuronal function in conditions associated with synaptic loss. Maintenance of neural connections provides improved function in neurodegenerative diseases relative to untreated patients. Prevention of synaptic loss can include at least a measurable improvement, such as at least a 10% improvement in the number of synapses, at least a 20% improvement, at least a 50% improvement, or more, relative to a control lacking such treatment, over a period of more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or at least a week.

[0217] Preferably, the medicament of the present invention is administered at a dose that reduces synaptic loss while minimizing any side effects. It is expected that the composition will be obtained and used for in vivo use under the guidance of a physician. The dosage of the therapeutic preparation will vary widely depending on the nature of the disease, the frequency of administration, the mode of administration, the clearance of the medicament from the host, etc.

[0218] The effective amount of a therapeutic composition to be administered to a particular patient will depend on a variety of factors, several of which will vary from patient to patient. Using ordinary skills, a competent clinician will be able to adjust the dosage of a particular therapeutic composition or imaging composition during routine clinical trials.

[0219] Therapeutic agents (e.g., complement inhibitors, gene expression activators, etc.) can be incorporated into a variety of preparations for therapeutic administration by combining with appropriate pharmaceutically acceptable carriers or diluents, and can be formulated into solid, semi-solid, liquid or gaseous forms of preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. As such, administration of the compound can be achieved in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasal, intratracheal, etc. administration. The active agent can be systemic after administration, or can be localized by regional administration, use of intramural administration or use of implants, which act to maintain the active dose at the implant site.

[0220] For example, one strategy for drug delivery through the blood-brain barrier (BBB) ​​requires biochemical destruction of the BBB by osmotic means (such as mannitol or leukotrienes) or by the use of vasoactive substances (such as bradykinin). The possibility of using BBB opening to target specific agents is also an option. When the composition is administered by intravascular injection, the BBB disruptor can be co-administered with the therapeutic composition of the present invention. Other strategies for crossing the BBB may require the use of endogenous transport systems, including carrier-mediated transporters (such as glucose and amino acid carriers), receptor-mediated transcytosis of insulin or transferrin, and active efflux transporters (such as p-glycoprotein). Active transport moieties can also be conjugated to therapeutic compounds or imaging compounds for use in the present invention to facilitate transport across the epithelial wall of the blood vessel. Alternatively, drug delivery after the BBB is by direct intrathecal delivery of therapeutic agents or imaging agents to the skull, such as through an Ommaya reservoir.

[0221] Depending on the desired formulation, the pharmaceutical composition may include a pharmaceutically acceptable non-toxic carrier of a diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. 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. In addition, the pharmaceutical composition or formulation may include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, and the like. The composition may also include additional substances to approximate physiological conditions, such as pH adjusters and buffers, toxicity regulators, wetting agents, and detergents.

[0222] The composition may also include any of a variety of stabilizers, such as antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide may be compounded 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 compounding agents include sulfates, gluconates, citrates, and phosphates. The polypeptides of the composition may also be compounded with molecules that enhance their in vivo properties. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0223] Further guidance on formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of drug delivery methods, see Langer, Science 249: 1527-1533 (1990).

[0224] The pharmaceutical compositions can be administered for prophylactic treatment and / or therapeutic treatment. The toxicity and therapeutic efficacy of the active ingredients can be determined in cell cultures and / or experimental animals according to standard pharmaceutical procedures, including, for example, determining the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is 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 50 / ED 50 Compounds that exhibit large therapeutic indices are preferred.

[0225] The data obtained from cell culture and / or animal studies can be used in formulating a range of dosage for use in humans. The dose of the active ingredient is generally arranged to contain the ED with low toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0226] The pharmaceutical compositions described herein can be administered in a variety of different ways. Examples include administering compositions containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.

[0227] For oral administration, active ingredients can be used in solid dosage forms (such as capsules, tablets and powders), or in liquid dosage forms (such as elixirs, syrups and suspensions). One or more active components can be encapsulated in gelatin capsules 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). The examples of additional inactive ingredients that can be added to provide the desired color, taste, stability, buffering capacity, dispersibility or other known desired 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 into sustained release products to provide continuous release of medicines in a few hours. Compressed tablets can be coated with sugar coating or film to cover any unpleasant taste and protect tablets from the influence of air, or can be coated with enteric coating for selective decomposition in the gastrointestinal tract. Liquid dosage forms for oral administration may contain coloring and flavoring to increase patient acceptance.

[0228] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickening agents, stabilizers and preservatives.

[0229] The components used to prepare the pharmaceutical composition are preferably high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, usually at least analytical grade, and more typically at least pharmaceutical grade). In addition, compositions intended for in vivo use are generally sterile. In this sense, a given compound must be synthesized before use, and the resulting product is generally substantially free of any possible poisons, particularly any endotoxins, which may be present in the synthesis or purification process. Compositions for parenteral administration are also sterile, substantially isotonic and prepared under GMP conditions.

[0230] The compositions of the present invention can be administered using any medically appropriate procedure, such as intravascular (intravenous, intraarterial, intracapillary) administration, injection into the cerebrospinal fluid, intracavity or direct injection into the brain. Intrathecal administration can be implemented using an Ommaya reservoir according to known techniques. (F. Balis et al., Am J. Pediatr. Hematol. Oncol. 11, 74, 76 (1989)).

[0231] In cases where the therapeutic agent is administered locally in the brain, one method for administering the therapeutic composition of the invention is by deposition into or near the site by any suitable technique, such as by direct injection (with stereotactic assistance by a syringe if necessary) or by placing the tip of the Ommaya reservoir into a cavity or capsule for administration. Alternatively, a convection-enhanced delivery catheter can be implanted directly into the site, into a natural or surgically created capsule, or into normal brain matter. Such convection-enhanced drug composition delivery devices greatly improve the diffusion of the composition throughout the brain matter. The implanted catheters of these delivery devices utilize high-flow microinfusion (with flow rates ranging from about 0.5 μl / min to 15.0 μl / min) rather than diffuse flow to deliver the therapeutic composition to the brain and / or tumor mass. Such a device is described in U.S. Pat. No. 5,720,720, which is incorporated herein by reference in its entirety.

[0232] The effective amount of the therapeutic composition to be administered to a particular patient will depend on a variety of factors, several of which will vary from patient to patient. A competent clinician will be able to determine the effective amount of the therapeutic agent to be administered to a patient. The dosage of the agent will depend on the treatment, the route of administration, the nature of the therapeutic agent, the patient's sensitivity to the therapeutic agent, etc. 50 Animal data and other information, clinicians can determine the maximum safe dose of individuals according to the route of administration. Using ordinary skills, competent clinicians will be able to optimize the dosage of a specific therapeutic composition during routine clinical trials. The composition can be administered to the subject in a series of more than one administration. For therapeutic compositions, sometimes regular regular administration will be necessary, or it may be desirable. The treatment regimen will change with the dosage; for example, some medicaments can be taken for a long period of time on the basis of once a day or twice a day, while more selective medicaments can be administered in a more determined time course, such as one day, two days, three days or more days, one week or more weeks, one month or more months, etc., once a day, twice a day, half a week, once a week, etc.

[0233] The formulation can be optimized for retention and stability in the brain. When the agent is administered into the cranial compartment, it is desirable that the agent is retained in the compartment and does not diffuse or otherwise cross the blood-brain barrier. Stabilization techniques include cross-linking, polymerizing or being connected to a group (such as polyethylene glycol, polyacrylamide, neutral protein carrier, etc.) to achieve an increase in molecular weight.

[0234] Other strategies for increasing retention include embedding the agent in a biodegradable or bioerodible implant. The release rate of the therapeutically active agent is controlled by the transport rate through the polymer matrix, and the biodegradation of the implant. The transport of the drug through the polymer barrier will also be affected by the following factors: compound solubility; polymer hydrophilicity; polymer cross-linking degree; polymer swelling when absorbing water to make the polymer barrier more permeable to the drug; implant geometry, etc. The implant is sized and shaped to the area selected as the implant site. The implant can be a particle, a sheet, a patch, a plate, a fiber, a microcapsule, etc., and can have any size and shape compatible with the selected insertion site.

[0235] Implants can be monolithic, i.e., have the active agent uniformly distributed throughout the polymer matrix, or encapsulated, wherein a reservoir of the active agent is encapsulated by the polymer matrix. The choice of polymer composition to be employed will vary with the site of administration, the desired treatment period, patient tolerance, the nature of the disease to be treated, etc. The properties of the polymer will include biodegradability at the implantation site, compatibility with the agent of interest, ease of encapsulation, and half-life in a physiological environment.

[0236] Biodegradable polymer compositions that can be used can be organic esters or ethers that produce physiologically acceptable degradation products when they degrade, including monomers. Anhydrides, amides, orthoesters, etc., can play a role by themselves or in combination with other monomers. The polymer will be a condensation polymer. The polymer can be cross-linked or uncross-linked. Of particular interest are polymers (homopolymers or copolymers) of hydroxy aliphatic carboxylic acids, and polysaccharides. Among the polyesters of interest are polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone and combinations thereof. By using L-lactate or D-lactate, slowly degradable polymers are obtained, while racemates greatly increase degradation. Of particular interest are copolymers of glycolic acid and lactic acid, wherein the biodegradation rate is controlled by the ratio of glycolic acid to lactic acid. The fastest degrading copolymers have approximately equal amounts of glycolic acid and lactic acid, wherein any homopolymer is more resistant to degradation. The ratio of glycolic acid to lactic acid will also affect the fragility of the implant, wherein for larger geometries, more elastic implants are desired. Calcium alginate and functionalized cellulose (particularly carboxymethyl cellulose esters, characterized in being insoluble in water, having a molecular weight of about 5 kD to 500 kD, etc.) are in the polysaccharide of interest. Biodegradable hydrogels can also be used in the implants of the subject invention. Hydrogels are typically copolymer materials characterized by the ability to absorb liquids. Exemplary biodegradable hydrogels that can be used are described in Heller in: Hydrogels in Medicine and Pharmacy, NA Peppes ed., Vol. III, CRC Press, Boca Raton, Fla., 1987, pp 137-149.

[0237] The present invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. Associated with such one or more containers may be a notice in a form prescribed by a governmental agency regulating the manufacture, use or sale of a drug or biological product, indicating that the manufacture, use or sale agency is approved for human administration.

[0238] Compound screening

[0239] In some embodiments, candidate agents are screened according to the ability to regulate synaptic loss, and the agents may include candidate complement inhibitors, variants, fragments, mimetics, agonists, and antagonists. Such compound screening can be performed using in vitro models, genetically modified cells or animals, or purified proteins. Various assays can be used for this purpose. In one embodiment, compounds predicted to be antagonists or agonists of complement (including specific complement proteins (e.g., C1q) and complement activation signals (e.g., amyloid, APP), etc.) are tested in an in vitro culture system as described below.

[0240] For example, candidate agents can be identified by known pharmacology, by structural analysis, by using computer-based rational drug design, by binding assays, etc. A variety of in vitro models can be used to determine whether a compound binds to complement, or otherwise affects complement activity. Such candidate compounds are used to contact neurons in an environment that is permissive for synaptic loss. Such compounds can also be tested for their effect on synaptic loss in in vivo models.

[0241] Screening can also be performed for molecules produced by astrocytes (e.g., immature astrocytes) that induce C1q expression in neurons. In such an assay, a co-culture of neurons and astrocytes is evaluated for production or expression of a molecule that induces C1q expression. For example, a blocking antibody can be added to the culture medium to determine the effect on the induction of C1q expression in neurons.

[0242] Synaptic loss is quantified by administering a candidate agent to neurons in the culture medium and determining the presence of synapses in the absence or presence of an agent. In one embodiment of the invention, neurons are, for example, primary cultures of retinal ganglion cells (RGC). Purified RGC colonies are obtained by conventional methods, such as sequential immunopanning. Cells are cultured in a suitable medium, which will generally include appropriate growth factors, such as CNTF; BDNF, etc. Neuronal cells (e.g., RCG) are cultured for a period of time sufficient to allow strong protrusions to grow, and then cultured with candidate agents for a period of about 1 day to 1 week. In some embodiments, neurons are cultured on living astrocyte feeders to induce signal conduction of synaptic loss. Methods for culturing astrocyte feeder layers are known in the art. For example, cortical glial cells can be deposited in a medium that does not allow neurons to survive, and non-adherent cells are removed.

[0243] As known in the art, for quantification of synapses, the culture medium is fixed, blocked and washed, then stained with antibody-specific synaptic proteins (e.g., synaptotagmin, etc.), and developed with appropriate reagents. Staining analysis can be performed with a microscope. In one embodiment, the digital image of the fluorescent emission is adjusted with a camera and image acquisition software to remove the unused portion of the pixel value range, and the pixel values ​​used are adjusted to utilize the entire pixel value range. The corresponding channel images can be merged to produce a color (RGB) image containing two single channel images as separate color channels. Co-localization points can be identified using a rolling ball background difference algorithm to remove low-frequency background from each image channel. The number, average area, average minimum and maximum pixel intensity, and average pixel intensity of all synaptotagmins, PSD-95, and co-localization points in the image are recorded and stored to disk for analysis.

[0244] As used herein, the term "agent" describes any molecule, e.g., a protein or drug, that has the ability to modulate synaptic loss, particularly through the complement pathway. In a preferred embodiment, the agent is an anti-C1q antibody Fab fragment. Candidate agents also include gene components that inhibit C1q expression (e.g., antisense and RNAi molecules) and constructs encoding complement inhibitors (e.g., CD59), etc. Although candidate agents are typically organic molecules (including small organic compounds with a molecular weight greater than 50 Daltons and less than about 2500 Daltons), they include many chemical classes. Candidate agents include functional groups necessary for structural interactions with proteins (particularly hydrogen bonding), and typically include at least one amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. Candidate agents typically include cyclic carbon structures or heterocyclic structures and / or aromatic structures or polycyclic aromatic structures substituted with one or more of the above functional groups. Candidate agents are also found in biomolecules including peptides, carbohydrates, fatty acids, steroids, purines, pyrimidines, and derivatives, structural analogs, or combinations thereof. Typically, multiple assay mixtures are run in parallel at different agent concentrations to obtain differential responses to different concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the detection level.

[0245] Condition of interest

[0246] As used herein, by "neurological" or "cognitive" function, it is meant that the reduction of synapses in the brain enhances the patient's thinking ability, function, etc. As used herein, the term "subject" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class of mammals: humans, non-human primates (such as chimpanzees and other apes and monkeys); livestock (such as cattle, horses, sheep, goats, pigs); domestic animals (such as rabbits, dogs and cats); laboratory animals (including rodents, such as rats, mice and guinea pigs), etc. The term does not indicate a particular age or sex.

[0247] For the method of inhibiting synaptic loss of the present application, the conditions of interest include various neurodegenerative conditions, for example, Alzheimer's disease, Down syndrome, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, myotonic dystrophy, glaucoma, Parkinson's disease, etc. Complement-related eye conditions are also included, such as choroidal neovascularization (CNV) and age-related macular degeneration (AMD). Such conditions benefit from the administration of complement inhibitors, which include inhibitors of C1q, which allow the maintenance of synapses or reduced synaptic loss. In some cases, in the case of existing neuronal loss, it is also possible to expect enhanced neurogenesis, such as by increasing the administration of neurogenesis agents or regimens, transplantation of neuronal progenitor cells, etc. Agents that enhance synaptic generation (such as thrombospondin) can also be administered.

[0248] Other conditions of interest are autoimmune conditions or complement-mediated ocular conditions.

[0249] Age-related macular degeneration (AMD) is the main cause of blindness in the elderly around the world.AMD is characterized by the progressive loss of central vision caused by degenerative changes and neovascular changes in the macula (highly specialized area of ​​the eye retina responsible for fine visual acuity). Recent estimates show that 1,400 million people are blind or have severe visual impairment due to AMD. The disease has a great impact on the physical and mental health of the elderly population and their families, and is becoming a major public health burden. However, new discovery begins to provide the related cell activity, genetic factors and biochemical processes relevant to early AMD and how AMD becomes a clearer picture of the eye condition related to complement. There are two types of AMD, non-exudative (dry) AMD and exudative (wet) AMD. Dry or non-exudative forms relate to atrophic changes and hypertrophic changes in the retinal pigment epithelial cells (RPE) below the central retina (macula) and deposits (drusen) on the RPE. Patients suffering from non-exudative AMD can develop into wet or exudative forms of AMD, wherein the abnormal blood vessels referred to as choroidal neovascular membrane (CNVM) grow under the retina, exudate fluid and blood, and eventually cause blinding discoid scars in and under the retina. Non-exudative AMD is more common, and it is usually a precursor of exudative AMD. The performance of non-exudative AMD is different; hard drusen, soft drusen, RPE map-like atrophy and pigment agglutination can occur. Complement components are deposited on RPE in the early stages of AMD, and are the main components of drusen.

[0250] Alzheimer's disease is a progressive, inexorable loss of cognitive function associated with an excess of senile plaques in the cerebral cortex and subcortical gray matter, which also contain beta-amyloid and neurofibrillary tangles composed of tau protein. The common form affects people >60 years of age, and its incidence increases with age. It accounts for more than 65% of dementia in the elderly.

[0251] The cause of Alzheimer's disease is unknown. In about 15% to 20% of cases, the disease runs in families. The remainder are so-called sporadic cases, with some genetic determinants. In most early-onset cases and some late-onset cases, the disease has an autosomal dominant inheritance pattern, but with variable penetrance in old age. Environmental factors are the focus of active investigation.

[0252] During the course of the disease, synapses and eventually neurons are lost in the cerebral cortex, hippocampus and subcortical structures (including selective cell loss in the nucleus basalis of Meynert), locus coeruleus and nucleus raphae dorsalis. Cerebral glucose use and perfusion are reduced in some areas of the brain (in the early stages of the disease, the parietal lobe and temporal cortices, in the late stages of the disease, the prefrontal cortex). Neuroinflammatory plaques or senile plaques (composed of neurites, astrocytes and glial cells around the amyloid nucleus) and neurofibrillary tangles (composed of paired spiral filaments) play a role in the pathogenesis of Alzheimer's disease. Senile plaques and neurofibrillary tangles appear with normal aging, but they are more prevalent in people with Alzheimer's disease.

[0253] Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, always fatal neurological disease that attacks motor neurons. Signs of muscle weakness and atrophy and anterior horn cell dysfunction are initially observed most often in the hands and less often in the feet. The site of onset is random, and progression is asymmetric. Painful cramps are common and may precede weakness. Patients rarely survive 30 years, 50% die within 3 years of onset, 20% survive 5 years, and 10% survive 10 years. Diagnostic features include onset during middle or late adult life, and progressive, widespread motor involvement without sensory abnormalities. Until the late stages of the disease, the velocity of nerve conduction is normal.

[0254] Reductions in cell body area, synapse number, and total synaptic length have been reported even in apparently normal neurons of ALS patients. It has been proposed that when the plasticity of active zones reaches its limit, continued synaptic loss may lead to dysfunction. Preventing synaptic loss may preserve neuronal function in these patients.

[0255] Down syndrome is a chromosomal disorder that usually causes mental retardation (characteristic facies) and many other typical features, including microcephaly and short stature. In about 95% of cases, an extra entire chromosome 21 is present. At autopsy, the brains of adults with Down syndrome show typical microscopic findings of Alzheimer's disease, and many also develop associated clinical signs.

[0256] Parkinson's disease is an idiopathic, slowly progressing degenerative CNS disorder characterized by slow and reduced activity, muscle stiffness, resting tremor and postural instability. In primary Parkinson's disease, the neurons of the substantia nigra, locus coeruleus and other brainstem dopaminergic cell groups are lost. The cause of disease is unknown. The loss of substantia nigra neurons (which project to the caudate nucleus and shell) causes the depletion of the neurotransmitter dopamine in these regions. Onset is usually after 40 years old, in older age groups, with an increased incidence rate.

[0257] Secondary parkinsonism is caused by loss or disruption of dopamine function in the basal ganglia produced by other idiopathic degenerative diseases, drugs, or exotoxins. The most common cause of secondary parkinsonism is the ingestion of neuroleptic drugs or reserpine, which produce parkinsonism by blocking dopamine receptors. Less common causes include carbon monoxide or manganese poisoning, hydrocephalus, structural lesions (tumors, infarctions affecting the midbrain or basal ganglia), subdural hematomas, and degenerative disorders (including striatonigral degeneration).

[0258] Myotonic dystrophy is an autosomal dominant multisystem disorder characterized by malnutritional muscle weakness and myotonia. The molecular defect is an amplified trinucleotide (CTG) repeat sequence in the 3' untranslated region of the myotonic protein kinase gene on chromosome 19q. Symptoms can occur at any age, and the clinical severity range is wide. Myotonia is significant in the hand muscles, and ptosis is common even in mild cases. In severe cases, significant peripheral muscle weakness occurs, often accompanied by cataracts, premature baldness, axe-shaped facies, heart rate disorders, testicular atrophy and endocrine abnormalities (e.g., diabetes). Mental retardation is common. Severely affected people die in their 50s.

[0259] Glaucoma is a common neurodegenerative disease that affects retinal ganglion cells (RGC). Huge efforts are being spent to determine how RGC die in glaucoma. Evidence supports the existence of a partitioned degeneration program in synapses and dendrites (comprising RGCs). Recent data from in vitro studies and from genetic mouse models of glaucoma show that molecular differences in degenerative pathways form the basis of the destruction of RGC soma and RGC axons. In a variety of neurodegenerative diseases, axons, dendrites, and synapses often degenerate completely before cell death, and increasing evidence shows that this is important for the generation of clinical symptoms and signs.

[0260] Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by the development of abnormal mood, behavior, and mental abnormalities; loss of previously acquired intellectual or cognitive functions; and abnormal movements (dyskinesias). Classic symptoms of HD include the development of chorea or spontaneous, rapid, irregular, spastic movements that can affect the face, arms, legs, or trunk, and a gradual loss of thought processing and acquired intellectual abilities (dementia). There may be impairments in memory, abstract thinking, and judgment; false perceptions of time, place, or identity (disorientation); increased anxiety; and personality changes (schizotypal personality disorder). Although symptoms usually become apparent during the fourth or fifth decade of life, the age of onset is variable and ranges from early childhood to late adulthood (e.g., 70s or 80s).

[0261] HD is passed down in families as an autosomal dominant trait. The disorder occurs as a result of an abnormally long sequence, or "repeat," of instructions encoded within a gene on chromosome 4 (4p16.3). The progressive loss of nervous system function associated with HD results from the loss of neurons in certain areas of the brain, including the basal ganglia and cerebral cortex.

[0262] Multiple sclerosis is characterized by remissions and recurrent exacerbations of a wide variety of symptoms and signs of CNS dysfunction. The most common symptoms are paresthesias in one or more limbs, in the trunk, or on one side of the face; weakness or clumsiness in the legs or hands; or visual disturbances, for example, partial blindness or pain in one eye (retrobulbar neuritis), blurred vision, or dark spots. Other common early symptoms are ophthalmogenic palsies causing double vision (diplopia), transient weakness in one or more limbs, mild stiffness or abnormal fatigability of the limbs, minor gait disturbances, difficulty controlling bladder, vertigo, and mild mood disorders; all show scattered CNS involvement and often occur months or years before the disease is confirmed. Overheating can aggravate symptoms and signs.

[0263] The course of the disease is highly variable, unpredictable, and, in most patients, intermittent. Initially, episodes of remission may be separated by months or years, particularly when the disease begins as retrobulbar neuritis. However, some patients have frequent episodes and are rapidly incapacitated; in a few, the course may be rapid.

[0264] The method of the present invention can be used in combination with cell or tissue transplantation of the central nervous system, wherein such transplant comprises neural progenitor cells, such as neural progenitor cells found in fetal tissue, neural stem cells, embryonic stem cells or other cells and tissues expected to be used for neural repair or enhancement. Neural stem cells / progenitor cells have been described in the art, and their use in a variety of therapeutic regimens has been widely discussed. For example, among others, U.S. Pat. No. 6,638,501 (Bjornson et al.); U.S. Pat. No. 6,541,255 (Snyder et al.); U.S. Pat. No. 6,498,018 (Carpenter); U.S. Patent Application 20020012903 (Goldman et al.); Palmer et al. (2001) Nature 411(6833):42-3; Palmer et al. (1997) Mol Cell Neurosci. 8(6):389-404; Svendsen et al. (1997) Exp. Neurol. 148(1):135-46 and Shihabuddin (1999) Mol Med Today. 5(11):474-80; each of the above is hereby incorporated by reference into this document.

[0265] Neural stem cells and precursor cells can participate in normal development, including migration to disseminated CNS regions along established migration pathways, differentiation into a variety of developmentally appropriate and regionally appropriate cell types in response to microenvironmental signals, and non-destructive, non-tumorigenic dissemination with host progenitors and their progeny. Human NSCs are able to express foreign transgenes in vivo in these disseminated locations. As such, these cells are used in the treatment of a wide variety of conditions, including traumatic injuries 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, etc. Through synaptic loss enhancers, the functional connectivity of neurons is enhanced, providing improved clinical outcomes.

[0266] Pharmaceutical composition

[0267] The antibody Fab fragments of the present invention can be administered in the form of a pharmaceutical composition for the treatment of complement-related ocular conditions.

[0268] The therapeutic preparation of the antibody Fab fragment of the present invention is prepared for storage (in the form of a lyophilized preparation or an aqueous solution) by mixing the antibody Fab fragment having the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.

[1980] ). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations employed, and include buffers (such as phosphates, citrates and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight The present invention can be a polypeptide having a molecular weight (less than about 10 residues); a protein such as serum albumin, gelatin or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, histidine, arginine or lysine; a monosaccharide, a disaccharide and other carbohydrates including glucose, mannose or dextrin; a chelating agent such as EDTA; a sugar such as sucrose, mannitol, trehalose or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a zinc protein complex); and / or a nonionic surfactant such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0269] Lipofection or liposomes can also be used to deliver antibody Fab fragments into cells, with the smallest fragment that specifically binds to the binding domain of the target being preferred.

[0270] Antibody Fab fragments can also be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules) prepared, for example, by coacervation techniques or by interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0271] Preparations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0272] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibody Fab fragments, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid gamma ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT TM (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 more than 100 days, certain hydrogels release proteins for shorter time periods.

[0273] The compounds of the present invention for the prevention or treatment of ocular diseases or conditions are usually administered by ocular injection, intraocular injection and / or intravitreal injection. Other methods of administration are also used, including, but not limited to, topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal and intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0274] Preparations for ocular administration, intraocular administration or intravitreal administration can be prepared by methods known in the art and using ingredients known in the art. The main requirement for effective treatment is appropriate penetration through the eye. Different from the diseases in the front of the eye (wherein the drug can be delivered topically), retinal diseases require a more site-specific method. Eye drops and ointments rarely penetrate the back of the eye, and the blood-ocular barrier hinders systemically administered drugs from penetrating into the eye tissue. Therefore, the selection of drug delivery methods commonly used for the treatment of retinal diseases (such as AMD and CNV) is direct intravitreal injection. Usually, according to the patient's condition and the properties and half-life of the drug delivered, intravitreal injection is repeated at intervals of a certain time. For intraocular (e.g., intravitreal) penetration, molecules of smaller size are usually preferred.

[0275] The therapeutic efficacy of complement-related eye conditions (such as AMD or CNV) can be measured by various endpoints commonly used in evaluating intraocular diseases.For example, vision loss can be assessed.Can be by (but not limited to) for example, measure the average change of best corrected visual acuity (BCVA) from baseline to the expected time point (for example, wherein BCVA is based on early treatment diabetic retinopathy study (ETDRS) visual acuity table and the determination at the test distance of 4 meters), measure compared to baseline, lose the ratio of the subject of the visual acuity less than 15 letters at the expected time point, measure compared to baseline, obtain the ratio of the subject of the visual acuity more than or equal to 15 letters at the expected time point, measure at the expected time point with 20 / 2000 or worse visual acuity Snellen equivalent (Snellen equivalent) subject's ratio, measure NEI visual function questionnaire, measure the size of CNV at the expected time point and the leakage of CNV (for example, by fluorescein angiography) etc. and evaluate vision loss. An ocular assessment may be completed, for example, including, but not limited to, for example, performing an eye examination, measuring intraocular pressure, assessing visual acuity, measuring slit lamp pressure, assessing intraocular inflammation, etc.

[0276] Nucleic acids, vectors and host cells

[0277] The antibody Fab fragments of the present disclosure can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, an isolated nucleic acid having a nucleotide sequence encoding any of the antibody Fab fragments of the present disclosure is provided. Such a nucleic acid can encode a V fragment containing an anti-C1q antibody. L / C L The amino acid sequence of the anti-C1q antibody and / or V H / C H 1 amino acid sequence. In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. In some embodiments, host cells containing such nucleic acids are also provided. In some embodiments, the host cells contain (e.g., have been transduced with the following vectors): (1) a vector containing a nucleic acid encoding a V sequence containing an antibody L / C L The amino acid sequence and V containing antibody H / C H 1, or (2) a first vector containing a nucleic acid (the nucleic acid encoding the V L / C Land a second vector containing a nucleic acid (the nucleic acid encoding the V H / C H 1). In some embodiments, the host cell is eukaryotic, for example, a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a Y0, NS0, Sp20 cell).

[0278] Methods for making anti-C1q antibody Fab fragments of the present disclosure are provided. In some embodiments, the method comprises culturing a host cell of the present disclosure containing a nucleic acid encoding an anti-C1q antibody Fab fragment under conditions suitable for expression of the antibody Fab fragment. In some embodiments, the antibody Fab fragment is subsequently recovered from the host cell (or host cell culture medium).

[0279] For the recombinant production of the humanized anti-C1q antibody Fab fragments of the present disclosure, nucleic acids encoding the anti-C1q antibody Fab fragments are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0280] Suitable vectors containing nucleic acid sequences encoding any anti-C1q antibody Fab fragment of the present disclosure, or fragment polypeptides thereof (including antibodies) described herein include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the cloning vector selected can vary depending on the host cell intended for use, useful cloning vectors generally have the ability to replicate themselves, can have a single target for a specific restriction endonuclease, and / or can carry a marker gene that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, for example, pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors (such as pSA3 and pAT28). These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

[0281] An expression vector is generally a replicable polynucleotide construct containing a nucleic acid of the present disclosure. An expression vector can be replicated in a host cell as an episome or as an integrated part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenovirus, adeno-associated virus, retrovirus, cosmid, and one or more expression vectors disclosed in PCT Publication No. WO 87 / 04462. 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 transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as ribosome binding sites, translation start sites, and stop codons, are generally also required.

[0282] The vector containing the nucleic acid of interest can be introduced into the host cell by any of a variety of appropriate means, including electroporation, transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (for example, in the case where the vector is an infectious pathogen such as vaccinia virus). The choice of the vector or polynucleotide to introduce will generally depend on the characteristics of the host cell. In some embodiments, the vector contains a nucleic acid containing one or more amino acid sequences encoding an anti-C1q antibody of the present disclosure.

[0283] Suitable host cells for cloning or expressing antibody Fab fragment-encoding vectors include prokaryotes or eukaryotic cells. For example, the anti-C1q antibody Fab fragments of the present disclosure can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria (e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 describing expression of antibody fragments in E. coli; and Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254). After expression, the antibody can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

[0284] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to result in the production of antibodies with partially or fully human glycosylation patterns (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006)).

[0285] Suitable host cells for expressing glycosylated antibody Fab fragments can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Various baculovirus strains have been identified and can be used with insect cells, particularly for transfection of Spodopterafrugiperda cells. Plant cell culture media can also be used 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 PLANTIBODIESTM technology for producing antibodies in transgenic plants).

[0286] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (such as, for example, 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (such as, for example, TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); 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 hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (such as, for example, Mather et al., Annals NYAcad.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.USA 77:4216 (1980)); and myeloma cell lines (such as Y0, NS0 and Sp2 / 0). For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol.248 (BKCLo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003).

[0287] Combination therapy

[0288] The complement inhibitors of the present disclosure may be used, without limitation, in combination with any additional treatment (eg, immunosuppressive therapy) for any of the diseases disclosed herein (including autoimmune diseases and / or neurodegenerative diseases).

[0289] In some embodiments, the antibodies of the present disclosure are administered in combination with inhibitors of the alternative pathway of complement activation. Such inhibitors may include (but are not limited to): 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 factors or non-specific complement inhibitors such as nafamostat mesylate (FUTHAN; FUT-175), aprotinin, K-76 monocarboxylic acid (MX-1) and heparin (see, e.g., TEMollnes & M.Kirschfink, Molecular Immunology 43 (2006) 107–121). In some embodiments, the antibodies of the present disclosure are administered in combination with inhibitors of the interaction between autoantibodies and their own antigens. Such inhibitors may comprise a soluble form of a purified autoantigen, or an antigen mimetic such as a peptide or RNA derived mimotope (including a mimotope of the AQP4 antigen). Alternatively, such inhibitors may comprise a blocker that recognizes the autoantigen and prevents binding of the autoantibody without triggering the classical complement pathway. Such a blocker may comprise, for example, an autoantigen binding RNA aptamer or an antibody that lacks a functional C1q binding site in its Fc domain (e.g., a Fab fragment or an antibody that is otherwise engineered not to bind C1q).

[0290] Reagent test kit

[0291] The present invention also provides a kit containing the antibody Fab fragments of the present disclosure. The kit of the present invention comprises one or more containers comprising a purified anti-C1q antibody Fab fragment and instructions for use according to methods known in the art. Typically, these instructions include a description of administering the inhibitor to treat or diagnose a disease according to any method known in the art. The kit may also include a description of selecting an individual suitable for treatment based on identification of whether the individual has a disease and the stage of the disease.

[0292] The instructions typically include information about the dosage, dosage regimen, and route of administration for the intended treatment. The container can be a unit dose, a large package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kit of the present 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 storage disk or optical disk) are also acceptable.

[0293] The label or package insert indicates that the composition is used to treat a specific condition.Instructions may be provided for practicing any of the methods described herein.

[0294] The kit of the present invention is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc. It is also contemplated that the package is used in combination with a specific device, such as an inhaler, a nasal application device (e.g., a sprayer), or an infusion device (e.g., a micropump). The kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an inhibitor of the classical complement pathway. The container can also include a second pharmaceutically active agent.

[0295] The kit may optionally provide additional components, such as buffers and instructional information. Typically, the kit includes a container and a label or one or more package inserts on or associated with the container.

[0296] example

[0297] Example 1: C1q binding assay

[0298] Direct ELISA was used to assess the binding of Fab and full-length antibodies to human C1q (Figure 1). Briefly, black 96-well plates (#3925, Corning) were coated with human C1q (2 μg / mL) (Complement Technology) in bicarbonate buffer (pH 9.4) (Thermo Scientific) overnight at 4°C. The next day, the plates were washed three times with dPBS and blocked with 3% BSA / PBS for 1 hour at room temperature (RT). Serial dilutions of M1, M1-Fab were prepared in dPBS 0.3% BSA, 0.1% Tween buffer (50 μL / well). Subsequently, alkaline phosphatase (AP)-conjugated secondary antibodies were added to mouse kappa or mouse FC, human kappa and human FC (Jackson Immunoresearch) at 2000-4000 times, respectively. After an additional 1 hour incubation at RT, the plates were washed three times with dPBS 0.05% Tween and luminescence was developed with AP substrate (Thermo Scientific). Luminescence counts were measured using an Envision plate reader (Perkin Elmer). Counts were plotted as a function of concentration and the EC50 for C1q binding was derived using a 4-parameter logistic fit. As demonstrated in Figure 1, M1 Fab has the same binding affinity as the M1 full antibody. Figure 1B M1 whole antibody (bivalent) and M1 Fab (monovalent) are shown. It is expected that the M1 whole antibody will exhibit higher avidity binding to CIq than the M1 antibody Fab.

[0299] Example 2: Hemolysis

[0300] The ability of anti-C1q antibodies to inhibit RBC hemolysis was determined using sensitized sheep RBCs and normal human serum as sources of complement ( Figure 2 ). Briefly, antibodies (M1-full IgG, M1-Fab, humanized M1, and humanized M1-Fab) were titrated in the range of 10000 ng / mL to 10 ng / mL in 50× diluted normal human serum in GVB++ buffer (Complement Technology). Then, 50 μL of diluted serum was mixed with 50 μL of diluted sheep RBC (~100 million cells / mL) (Complement Technology). The samples were incubated at 37°C for 30 minutes. The plates were then centrifuged at 2000 rpm for 5 minutes, the supernatant was collected and the absorbance was read at 415 nm. Controls containing background hemolysis were run alone in GVB++ buffer without serum, and water was used to trigger maximum hemolysis. The untreated absorbance was subtracted from the background and normalized to the maximum hemolysis, and plotted as a function of concentration. The IC50 for inhibition of hemolysis was determined using a 4-parameter logistic fit. Figure 2 As demonstrated in , M1 Fab has the same functional potency as M1 whole antibody. Figure 1B ) ability, and it will exhibit higher energy efficiency due to the steric hindrance of other head groups due to its larger size.

[0301] Incorporated by Reference

[0302] Each of the patents, published patent applications, and non-patent references cited herein are incorporated by reference in their entirety.

[0303] Equivalent

[0304] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. An antibody Fab fragment that binds to C1q, the antibody Fab fragment comprising a heavy chain domain and a light chain domain, wherein the heavy chain domain consists of the amino acid sequence of SEQ ID NO: 1, and the light chain domain consists of the amino acid sequence of SEQ ID NO:

2. 2 . The antibody Fab fragment of claim 1 , wherein the antibody Fab fragment specifically binds to both human C1q and mouse C1q. 3 . The antibody Fab fragment of claim 1 , wherein the antibody Fab fragment binds to rat C1q, dog C1q, rhesus monkey C1q or cynomolgus monkey C1q, or a combination thereof. 4 . The antibody Fab fragment of claim 1 , wherein the antibody Fab fragment binds to human C1q, mouse C1q and / or rat C1q, and at least one of the following: dog C1q, rhesus monkey C1q and cynomolgus monkey C1q.

5. The antibody Fab fragment of claim 1 or 2, wherein the antibody Fab fragment has been engineered to increase brain penetrance.

6. The antibody Fab fragment according to claim 1 or 2, wherein the antibody Fab fragment has a dissociation constant (K) ranging from 10 pM to 20 pM, or from 1 pM to less than 10 pM for human C1q. D ).

7. The antibody Fab fragment according to claim 1 or 2, wherein the antibody Fab fragment has a dissociation constant (K) ranging from 1 pM to 200 pM for mouse C1q. D ). The antibody Fab fragment of claim 1 or 2, wherein the antibody Fab fragment specifically binds to C1q and neutralizes the biological activity of C1q.

9. The antibody Fab fragment of claim 8, wherein the biological activity is (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to phosphatidylserine, (5) C1q binding to pentraxin-3, (6) C1q binding to C-reactive protein (CRP), (7) C1q binding to globular C1q receptor (gC1qR), (8) C1q binding to complement receptor 1 (CR1), (9) C1q binding to β-amyloid protein, (10) C1q binding to calreticulin, (11) C1q binding to apoptotic cells, or (12) C1q binding to neural cell membrane components.

10. The antibody Fab fragment of claim 8, wherein the biological activity is (1) activation of the classical complement activation pathway, (2) activation of antibodies and complement-dependent cytotoxicity, (3) CH50 hemolysis, (4) synaptic loss, (5) B cell antibody production, (6) dendritic cell maturation, (7) T-cell proliferation, (8) cytokine production, (9) microglial activation, (10) Arthritis reaction, (11) phagocytosis of synapses or nerve terminals, or (12) activation of complement receptor 3 (CR3 / C3) expressing cells.

11. The antibody Fab fragment of claim 10, wherein CH50 hemolysis comprises human CH50 hemolysis, mouse CH50 hemolysis, rat CH50 hemolysis, dog CH50 hemolysis, rhesus monkey CH50 hemolysis, and / or cynomolgus monkey CH50 hemolysis.

12. The antibody Fab fragment of claim 11, wherein the antibody Fab fragment is capable of neutralizing CH50 hemolysis from at least 50% to at least 90%.

13. The antibody Fab fragment of claim 11, wherein 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.

14. The antibody Fab fragment of claim 10, wherein the antibody Fab fragment binds to C1q and inhibits a biological function with a binding stoichiometry ranging from less than 6:1 to 1:1, or less than 2:1 to 1:

1.

15. The antibody Fab fragment of claim 1 or 2, wherein the antibody Fab fragment has a shorter half-life compared to its corresponding full-length antibody.

16. A host cell expressing the antibody Fab fragment according to any one of claims 1 to 15.

17. A DNA encoding the heavy chain region and the light chain region of the antibody Fab fragment from any one of claims 1 to 15.

18. A cloning vector or expression vector comprising the DNA of claim 17.

19. The host cell of claim 16, comprising the cloning vector or expression vector of claim 18.

20. An isolated polynucleotide comprising a nucleic acid sequence encoding the antibody Fab fragment of any one of claims 1-15.

21. A method for producing the antibody Fab fragment of any one of claims 1-15, the method comprising culturing the host cell of claim 19 and isolating the antibody Fab fragment.

22. A pharmaceutical composition comprising the antibody Fab fragment according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

23. Use of an antibody Fab fragment selected from any one of claims 1 to 15 in the manufacture of a medicament for the treatment of Alzheimer's disease.

24. The use according to claim 23, wherein the drug is administered together with neural progenitor cells or a neurogenesis enhancer.

25. The use according to claim 23, wherein the antibody Fab fragment binds to C1q and inhibits complement activation.

26. Use of an antibody Fab fragment selected from any one of claims 1 to 15 in the manufacture of a medicament for the treatment of amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease or Huntington's disease.

27. The antibody Fab fragment selected from any one of claims 1-15 is used in the manufacture of diabetes, obesity, rheumatoid arthritis, acute respiratory distress syndrome, distal tissue damage after ischemia and reperfusion, dermatomyositis, pemphigus, lupus nephritis and glomerulonephritis and vasculitis caused by cardiopulmonary shunt, coronary endothelial dysfunction induced by cardiac arrest, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration, age-related macular degeneration, geographic atrophy, choroidal neovascularization, uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, eye Use of the drug in the treatment of neovascular disease, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica, central retinal vein occlusion, corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, anti-neutrophil cytoplasmic antibody vasculitis, Pursha retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome, asthma, aspiration pneumonia, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid or myositis.

28. Use of an antibody Fab fragment selected from any one of claims 1-15 in the manufacture of a medicament for the treatment of myasthenia gravis, type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, paraneoplastic syndrome, vasculitis, hypocomplementemic urticarial vasculitis, polymyalgia rheumatica, temporal arteritis, Wegener's granulomatosis, multiple sclerosis, Guillain-Barré syndrome, bullous pemphigoid or myositis.

29. A kit comprising the antibody Fab fragment of any one of claims 1-15 and a package insert comprising instructions for using the antibody Fab fragment to treat or prevent a disease in an individual in need of such treatment, wherein the disease is Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, Huntington's disease, diabetes, obesity, rheumatoid arthritis, acute respiratory distress syndrome, distal tissue damage after ischemia and reperfusion, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration, age-related macular degeneration, Geographic atrophy, choroidal neovascularization, uveitis, diabetic retinopathy, ischemic retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica, central retinal vein occlusion, corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, antineutrophil cytoplasmic antibody vasculitis, general Ersha retinopathy, Sjogren's dry eye, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome, asthma, aspiration pneumonia, myositis, type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, paraneoplastic syndromes, vasculitic disorders, hypocomplementemic urticarial vasculitis, polymyalgia rheumatica, or Wegener's granulomatosis.

30. Use of an antibody Fab fragment according to any one of claims 1 to 15 in the preparation of a kit for determining a subject's risk of developing a disease, wherein the antibody Fab fragment is coupled to a detectable marker; wherein the risk of developing a disease is characterized based on a comparison of the amount of C1q compared to a reference; and wherein the disease is Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, spinal cord injury; Muscular dystrophy, Down syndrome, Parkinson's disease, Huntington's disease, diabetes, obesity, rheumatoid arthritis, acute respiratory distress syndrome, distal tissue damage after ischemia and reperfusion, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration , age-related macular degeneration, geographic atrophy, choroidal neovascularization, uveitis, diabetic retinopathy, ischemic retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica, central retinal vein occlusion, corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, antineutrophil cytoplasmic antibodies Vasculitis, Pursha retinopathy, Sjogren's dry eye, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome, asthma, aspiration pneumonia, myositis, type 1 diabetes mellitus, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, paraneoplastic syndromes, vasculitic disorders, hypocomplementemic urticarial vasculitis, polymyalgia rheumatica, or Wegener's granulomatosis.

31. The use according to claim 30, wherein the detectable label comprises a nucleic acid, an oligonucleotide, an enzyme, a radioisotope, biotin or a fluorescent label.

32. The use according to claim 31, wherein the detectable marker is detected using imaging agents for X-ray, CT, MRI, ultrasound, PET and SPECT.

33. The use according to claim 31, wherein the fluorescent marker is selected from fluorescein, rhodamine, cyanine dye or BODIPY.

34. Use of an antibody Fab fragment selected from any one of claims 1 to 15 in the manufacture of a medicament for reducing the risk of developing a disease in a subject, wherein the antibody Fab fragment prevents or reduces the risk of developing the disease, and wherein the disease is Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, Huntington's disease, diabetes Diabetes, obesity, rheumatoid arthritis, acute respiratory distress syndrome, distal tissue damage after ischemia and reperfusion, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration, age-related macular degeneration, geographic atrophy , choroidal neovascularization, uveitis, diabetic retinopathy, ischemic retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica, central retinal vein occlusion, corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, antineutrophil cytoplasmic antibody vasculitis, Pursha Retinopathy, Sjogren's dry eye, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome, asthma, aspiration pneumonia, myositis, type 1 diabetes mellitus, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, paraneoplastic syndromes, vasculitic disorders, hypocomplementemic urticarial vasculitis, polymyalgia rheumatica, or Wegener's granulomatosis.

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