Compositions and methods for treating epilepsy

Abstract

The present disclosure relates generally to methods of preventing, reducing the risk of developing epilepsy, or treating epilepsy comprising administering to a subject an inhibitor of the classical complement pathway.

Description

[0001] Statement Regarding Federally Funded Research

[0002] This invention was made with government support under ARMY / MRMC Grant W81XWH-16-1-0576. The government has certain rights in this invention.

[0003] Related applications

[0004] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 020,245, filed May 5, 2020, which is hereby incorporated by reference in its entirety.

[0005] Sequence Listing

[0006] This application contains a sequence listing submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy was created on May 3, 2021, is named ANH-01025_SL.txt, and is 41,770 bytes in size. Background Art

[0007] Epilepsy is a common neurological condition and has been identified as one of the most prevalent neurological conditions characterized by nerve cell damage or loss. According to estimates by the World Health Organization, approximately 50 million people worldwide are affected by epilepsy, and nearly 80% of affected individuals live in developing countries. One in ten people will experience at least one epileptic seizure during their normal lifespan, and one-third of these individuals will develop epilepsy. While all age groups can be affected by epileptic seizures, the condition is most prevalent in the young and elderly. Epilepsy is one of the most common serious neurological conditions in the United States and often requires long-term treatment. Each year, 150,000 people in the United States are newly diagnosed with epilepsy.

[0008] Although antiepileptic drugs (ezogabine, pregabalin, levetiracetam, lamotrigine, topiramate, valproate, rufinamide, gabapentin, carbamazepine, clonazepam, oxcarbazepine, phenobarbital and phenytoin) are available recently, available treatment options are not enough to effectively prevent or treat the disease, and seizures are still difficult to completely eradicate; about one-third of patients still have uncontrollable seizures, and even a larger proportion suffers from at least one anticonvulsant-related side effect (such as mood changes, drowsiness or gait instability). In addition, although seizures represent the most significant feature of epilepsy, many epilepsy patients develop neurological or psychiatric diseases (memory or cognitive impairment, depression ...). For example, mesial temporal lobe epilepsy is usually accompanied by memory impairment, which may be due to hippocampal system damage and / or brain inflammation.

[0009] Although the treatment of epilepsy has advanced over the past decade, available treatment options focus on preventing seizures, and current drug therapies may not cure or even ameliorate the course of the disease. Currently available anti-epileptic drugs do not appear to be anti-epileptic. This may be due to the fact that current agents act in a mechanistically inappropriate manner to prevent disease progression. Therefore, there is a need in the art for new therapies to prevent and treat epilepsy. Summary of the Invention

[0010] The present disclosure generally relates to methods of preventing, reducing the risk of developing epilepsy, or treating epilepsy by inhibiting complement activation, such as by inhibiting the classical complement pathway.

[0011] The present disclosure generally relates to methods of preventing, reducing the risk of developing, or treating epilepsy (e.g., idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy) by inhibiting classical complement activation, e.g., by inhibiting complement factors Clq, Clr, or Cls, e.g., by administering antibodies, such as monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, antibody derivatives, etc., that bind one or more of these complement factors.

[0012] In some embodiments, the activity of complement factors such as C1q, C1r, or C1s is inhibited to block activation of the classical complement pathway and slow or prevent epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy). Inhibition of the classical complement pathway leaves the lectin and alternative complement pathways intact to perform their normal immune functions. Disclosed herein are methods related to neutralizing complement factors (e.g., C1q, C1r, or C1s) in epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy).

[0013] In one aspect, the present disclosure provides methods of preventing, reducing the risk of developing epilepsy, or treating epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy (e.g., temporal lobe epilepsy)) comprising administering to a subject an inhibitor of the classical complement pathway.

[0014] Also provided are a number of embodiments that can be applied to any aspect of the invention described herein. For example, in some embodiments, the inhibitor is administered within the first 4 weeks during or after an attack, within the first week during or after an attack, within 24 hours during or after an attack, or within 1, 2, 3, 4, 5, or 6 hours during or after an attack. In some embodiments, the inhibitor suppresses synaptic loss induced by an attack. In some embodiments, the inhibitor is administered to a patient suffering from traumatic brain injury, anoxic brain injury, brain infection, stroke, or a genetic syndrome. The brain infection may be encephalitis, meningitis, mesial temporal sclerosis, or a brain tumor. Epilepsy may be induced by traumatic brain injury, anoxic brain injury, brain infection, stroke, or a genetic syndrome. Epilepsy may be TBI-induced epilepsy. In some embodiments, the inhibitor suppresses synaptic loss induced by traumatic brain injury, anoxic brain injury, brain infection, stroke, or a genetic syndrome. In some embodiments, the inhibitor is administered during or within the first 4 weeks after the onset of a traumatic brain injury, anoxic brain injury, brain infection, or stroke; during or within the first week after the onset of a traumatic brain injury, anoxic brain injury, brain infection, or stroke; within 24 hours during or after the onset of a traumatic brain injury, anoxic brain injury, brain infection, or stroke; or within 1, 2, 3, 4, 5, or 6 hours during or after the onset of a traumatic brain injury, anoxic brain injury, brain infection, or stroke.

[0015] In some embodiments, the inhibitor of the classical complement pathway is a C1q inhibitor. For example, the C1q inhibitor can be an antibody, an aptamer, an antisense nucleic acid, or a gene editing agent. In some embodiments, the antibody is an anti-C1q antibody. In some embodiments, the anti-C1q antibody inhibits the interaction between C1q and autoantibodies, or between C1q and C1r, or between C1q and C1s. In some embodiments, the anti-C1q antibody promotes the clearance of C1q from the circulation or tissues. In some embodiments, the antibody is an anti-C1q antibody having a dissociation constant (KD) ranging from 100 nM to 0.005 nM or less than 0.005 nM. In some embodiments, the antibody is an anti-C1q antibody that binds C1q with a binding stoichiometry ranging from 20:1 to 1.0:1 or less than 1.0:1, binds C1q with a binding stoichiometry ranging from 6:1 to 1.0:1 or less than 1.0:1, or binds C1q with a binding stoichiometry ranging from 2.5:1 to 1.0:1 or less than 1.0:1. In some embodiments, the antibody specifically binds to and neutralizes a biological activity of C1q, such as (1) binding of C1q to autoantibodies, (2) binding of C1q to C1r, (3) binding of C1q to C1s, (4) binding of C1q to IgM, (5) binding of C1q to IgG, (6) binding of C1q to phosphatidylserine, (7) binding of C1q to pentraxin-3, (8) binding of C1q to C-reactive protein (CRP), (9) binding of C1q to globular C1q receptor (gC1qR), (10) binding of C1q to complement receptor 1 (CR1), (11) binding of C1q to β-amyloid, (12) binding of C1q to calreticulin, (13) binding of C1q to apoptotic cells, or (14) binding of C1q to B cells. Another example of 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) microglia activation, (10) immune complex formation, (11) phagocytosis of synapses or nerve terminals, (12) activation of complement receptor 3 (CR3 / C3) expressing cells, or (13) neuroinflammation. In some embodiments, CH50 hemolysis includes human, mouse, rat, dog, rhesus monkey, and / or cynomolgus monkey CH50 hemolysis. In some embodiments, the antibody is capable of neutralizing at least about 50% to at least about 90% of CH50 hemolysis, or neutralizing at least 50% of CH50 hemolysis, at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml, or less than 20 ng / ml.

[0016] The antibody can be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, an antibody fragment or an antibody derivative thereof, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a diabody or a single-chain antibody molecule. The antibody can be coupled to a labeling group, such as an optical label, a radioisotope, a radionuclide, an enzymatic group, a biotinyl group, a nucleic acid, an oligonucleotide, an enzyme or a fluorescent label.

[0017] In some preferred embodiments, the antibody comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acids of SEQ ID NO: 6, and HVR-L3 having the amino acids of SEQ ID NO: 7. Similarly, in some preferred embodiments, the antibody comprises a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acids of SEQ ID NO: 10, and HVR-H3 having the amino acids of SEQ ID NO: 11. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence that is at least about 95% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 35-38, and wherein the light chain variable domain comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acids of SEQ ID NO: 6, and HVR-L3 having the amino acids of SEQ ID NO: 7. In some embodiments, the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 35-38. In some embodiments, the antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 31-34, and wherein the heavy chain variable domain comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acids of SEQ ID NO: 10, and HVR-H3 having the amino acids of SEQ ID NO: 11. In some embodiments, the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 31-34. In other preferred embodiments, the antibody fragment comprises a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40.

[0018] In other embodiments, the inhibitor of the classical complement pathway is a C1r inhibitor. In some embodiments, the C1r inhibitor is an antibody, an aptamer, an antisense nucleic acid, or a gene editing agent. In some embodiments, the antibody is an anti-C1r antibody. In some embodiments, the anti-C1r antibody inhibits the interaction between C1r and C1q or between C1r and C1s, or wherein the anti-C1r antibody inhibits the catalytic activity of C1r, or it inhibits the processing of pro-C1r into an active protease. In some embodiments, the antibody is an anti-C1r antibody having a dissociation constant (KD) ranging from 100 nM to 0.005 nM or less. In some embodiments, the antibody is an anti-C1r antibody that binds to C1r with a binding stoichiometry ranging from 20:1 to 1.0:1 or less, ranging from 6:1 to 1.0:1 or less, or ranging from 2.5:1 to 1.0:1 or less. In some embodiments, the anti-C1r antibody promotes clearance of C1r from circulation or tissues.

[0019] In other embodiments, the inhibitor of the classical complement pathway is a C1s inhibitor. The C1s inhibitor can be an antibody, an aptamer, an antisense nucleic acid, or a gene editing agent. In some embodiments, the antibody is an anti-C1s antibody. In some embodiments, the anti-C1s antibody inhibits the interaction between C1s and C1q, or between C1s and C1r, or between C1s and C2 or C4, or wherein the anti-C1s antibody inhibits the catalytic activity of C1s, or it inhibits the processing of pro-C1s into an active protease, or it binds to an activated form of C1s. In some embodiments, the antibody is an anti-C1s antibody with a dissociation constant (KD) ranging from 100 nM to 0.005 nM or less than 0.005 nM. In some embodiments, the antibody is an anti-C1s antibody that binds C1s with a binding stoichiometry ranging from 20: 1 to 1.0: 1 or less, ranging from 6: 1 to 1.0: 1 or less, or ranging from 2.5: 1 to 1.0: 1 or less. In some embodiments, the anti-C1s antibody promotes clearance of C1s from circulation or tissues.

[0020] In other embodiments, the inhibitor of the classical complement pathway is an anti-C1 complex antibody, optionally wherein the anti-C1 complex antibody inhibits activation of C1r or C1s or prevents their ability to act on C2 or C4, for example, the anti-C1 complex antibody binds to a combined epitope within the C1 complex, wherein the combined epitope comprises amino acids from both C1q and C1s; amino acids from both C1q and C1r; amino acids from both C1r and C1s; or amino acids from each of C1q, C1r, and C1s. The antibody can be a monoclonal antibody. In some embodiments, the antibody inhibits cleavage of C4 without inhibiting cleavage of C2, or inhibits cleavage of C2 without inhibiting cleavage of C4.

[0021] In some embodiments, the antibody binds mammalian CIq, CIr, or CIs, or binds human CIq, CIr, or CIs. In some embodiments, the antibody binds mammalian CI complex.

[0022] In some embodiments, the antibody is a mouse antibody, a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv, and F(ab')2 fragments. In some embodiments, the antibody is a bispecific antibody that recognizes a first antigen and a second antigen. For example, the first antigen can be selected from C1q, C1r, and C1s, and the second antigen can be an antigen that promotes transport across the blood-brain barrier. The second antigen can be transferrin receptor (TR), insulin receptor (HIR), insulin growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, llama single domain antibody, TMEM 30 (A), protein transduction domain, TAT, Syn-B, transmembrane, polyarginine peptide, angiopoietin peptide, or ANG1005.

[0023] In some embodiments, the amount of the antibody that inhibits the classical complement activation pathway ranges from at least 30% to at least 99.9%. In some embodiments, the antibody inhibits the alternative complement activation pathway initiated by C1q binding. In some embodiments, the amount of the antibody that inhibits the alternative complement activation pathway ranges from at least 30% to at least 99.9%. In some embodiments, the antibody inhibits complement dependent cell-mediated cytotoxicity (CDCC), for example, the amount of the antibody that inhibits complement dependent cell-mediated cytotoxicity (CDCC) activation pathway ranges from at least 30% to at least 99.9%. In some embodiments, the antibody inhibits autoantibodies and complement dependent cell-mediated cytotoxicity (CDCC).

[0024] In some embodiments, the method further comprises administering a secondary antibody selected from an anti-C1q antibody, an anti-C1r antibody, and an anti-C1s antibody. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an inhibitor of antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an inhibitor of the classical complement activation pathway. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an inhibitor of the alternative complement activation pathway. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an inhibitor of the interaction between an autoantibody and its corresponding autoantigen.

[0025] In another aspect, a method for determining the risk of developing epilepsy in a subject due to traumatic brain injury, anoxic brain injury, brain infection, stroke, or a genetic syndrome is provided, comprising: (a) administering an anti-C1q, anti-C1r, or anti-C1s antibody to the subject, wherein the anti-C1q, anti-C1r, or anti-C1s antibody is conjugated to a detectable label; (b) detecting the detectable label to measure the amount or location of C1q, C1r, or C1s in the subject; and (c) comparing the amount or location of one or more of C1q, C1r, or C1s to a reference, wherein the risk of developing epilepsy is characterized based on the comparison of the amount or location of one or more of C1q, C1r, or C1s to the reference. In some embodiments, the brain infection is encephalitis, meningitis, mesial temporal sclerosis, or a brain tumor. The detectable label may comprise a nucleic acid, an oligonucleotide, an enzyme, a radioisotope, biotin, or a fluorescent label. In some embodiments, the antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv, and F(ab')2 fragments.

[0026] In some embodiments, an anti-C1q antibody comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acids of SEQ ID NO: 6, and HVR-L3 having the amino acids of SEQ ID NO: 7. In some embodiments, an anti-C1q antibody comprises a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acids of SEQ ID NO: 10, and HVR-H3 having the amino acids of SEQ ID NO: 11. In some embodiments, an anti-C1q antibody comprises a light chain variable domain comprising an amino acid sequence that is at least about 95% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 35-38, and wherein the light chain variable domain comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acids of SEQ ID NO: 6, and HVR-L3 having the amino acids of SEQ ID NO: 7. In some embodiments, the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 35-38. In some embodiments, the anti-C1q antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 31-34, and wherein the heavy chain variable domain comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acids of SEQ ID NO: 10, and HVR-H3 having the amino acids of SEQ ID NO: 11. In some embodiments, the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 31-34.

[0027] In some embodiments, epilepsy is idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy or symptomatic partial epilepsy (e.g., temporal lobe epilepsy). In some embodiments, symptomatic generalized epilepsy or symptomatic partial epilepsy is induced by traumatic brain injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1A-1E Depicts that three weeks after mTBI, the damaged cortex and functionally connected thalamus show chronic inflammation and neuronal loss. Figures 1A-1B Schematic diagram of a coronal mouse brain section showing the site and depth of controlled cortical impact ( Figure 1A ) and the locations of the S1 cortex and the nRT and VB thalamic regions ( Figure 1BThe impactor had a diameter of 3 mm and an impact depth of 0.8 mm, reaching the right somatosensory cortex. Figure 1C Representative coronal brain sections of mTBI mice stained for C1q are shown. Bilateral C1q expression in the hippocampus is typical of physiological conditions and is present in both sham-operated and mTBI mice. Figure 1D Shown are close-up images of S1 (top), VB, and nRT (middle), as well as confocal images of nRT (bottom), stained for C1q, the neuronal marker NeuN, the astrocyte marker GFAP, and the microglia / macrophage marker IBA1. The injury site in the right S1 cortex is marked with an asterisk. Arrows in nRT indicate the location of the confocal images. Scale bars, 300 μm (top / middle) and 20 μm (bottom). Figure 1E Quantification of the fluorescence ratio between ipsilateral and contralateral regions in sham-operated and mTBI mice is shown. Data represent all points from minimum to maximum, using the Mann-Whitney test and α = 0.05 (*p < 0.05, **p < 0.01). Analysis included five to seven mice per group (n = three sections per mouse, one image per region).

[0029] Figures 2A-2I Depicts that three weeks after mTBI, nRT ipsilateral to the injured cortex displays neuronal loss and altered IPSC and EPSC properties. Figures 2A-2C A high-power coronal image of the nRT is shown, showing the division of the "head", "body" and "tail" ( Figure 2A ), and quantification of neuronal counts across the ipsilateral nRT ( Figure 2B ) or quantification of neuronal counts per segment, normalized to the median value of the sham group ( Figure 2C Neuron count data represent mean ± SEM, using the Mann-Whitney test, α = 0.05 (*p < 0.05, **p < 0.01). The analysis included six mice per group (n = an average of three sections per mouse). Figures 2D-2E Spontaneous IPSC recordings from representative nRT neurons in sham-operated and mTBI mice are shown ( Figure 2D ), and the frequency and amplitude distributions of 13 posterior nRT neurons from four sham-operated mice and 22 posterior nRT neurons from six mTBI group mice ( Figure 2E IPSC data represent mean ± SEM and were analyzed using the Mann-Whitney test with α = 0.05 (*p < 0.05). Figures 2F-2G Spontaneous EPSC recordings from representative nRT neurons in sham-operated and mTBI mice are shown ( Figure 2F ), and the frequency and amplitude distributions of 11 posterior nRT neurons from six sham-operated mice and nine posterior nRT neurons from seven mTBI mice ( Figure 2G ). Insets show average EPSC traces of individual nRT neurons from sham-operated and mTBI mice, plotted on the same scale. EPSC data represent mean ± SEM analyzed by Mann-Whitney test, α = 0.05 (*p < 0.05). Figure 2H Representative images of coronal brain sections from Thy1-GCaMP6f mice with sham surgery (left panel) and mTBI (right panel) are shown (the injury site is marked with an asterisk). The lower panel shows projection terminals from the cortex to the VB and nRT. Scale bars, 1 mm (upper panel) and 500 μm (lower panel). A reduction in projection terminals from the cortex to the VB and nRT was observed in n = 6 mTBI mice (marked by arrows). Figure 2I Quantification of the Thy1-GCaMP fluorescence ratio between ipsilateral and contralateral regions in sham-operated and mTBI mice is shown. Data represent all points from minimum to maximum, using the Mann-Whitney test and α = 0.05 (*p < 0.05, **p < 0.01). Analysis included five sham-operated mice and six mTBI mice (n = three sections per mouse, one image per region).

[0030] Figures 3A-3H depicts single-nucleus RNA sequencing showing microglia as the source of C1q in the thalamus three weeks after mTBI. Figure 3A Depicted is a schematic diagram of a coronal brain section showing the anatomical location of thalamic tissue. Figure 3B 、 3C and 3E show the lineages through cell types ( Figure 3B ), nuclear C1qa expression ( Figure 3C ) or C4b expression ( Figure 3E ) Uniform Manifold Approximation and Projection (UMAP) projections of stained single nuclei (n=4,908 sham-operated cells, n=4,338 mTBI cells, after data cleaning). Figure 8A Lineage markers described in [ 15 ]. Coloring is done using fill rendering. Normalized expression ratios are shown above, with 0 = maximum, and each panel has a maximum value. Figure 3D and 3F Cluster 3 (Oligonucleotide 3, Figure 9E ) in the nuclei of microglia ( Figure 3D ) and C4b expression in oligodendrocyte nuclei ( Figure 3F ) were analyzed using the Wilcoxon rank-sum test (ns = not statistically significant). Two technical replicates were combined, representing nine sham-operated mice and ten mTBI mice. Each dot represents a single nucleus. Figure 3G and 3H It shows that C1qa( Figure 3E) and C4b( Figure 3H ) transcripts were quantified by RT-qPCR. Each dot represents the bulk RNA extracted from one replicate (n = two biobanks, each dot represents n = three technical replicates). The first replicate included five sham-operated mice and six mTBI mice, and the second replicate included four sham-operated mice and four mTBI mice.

[0031] Figures 4A-4C showed that three weeks after mTBI, anti-C1q antibodies reduced chronic inflammation and neuronal loss. Figure 4A and 4B Representative coronal brain sections from mice in the mTBI group are shown ( Figure 4A ) and close-up( Figure 4B Figure 3 S1 (upper panel), VB, and nRT (lower panel) of mTBI mice treated with anti-C1q antibodies and stained for C1q, NeuN, GFAP, and IBA1. The injury site in the right S1 cortex is marked with an asterisk. Scale bars, 1 mm (A), 500 μm (B). Figure 4C Quantification of nRT neuron counts and fluorescence ratios between ipsilateral and contralateral regions in control and antibody-treated sham-operated and mTBI mice is shown. Data represent all points from minimum to maximum, using the Mann-Whitney test and α = 0.05 (*p < 0.05, **p < 0.01). Analysis included six to eight mice per group (n = three sections per mouse, one image per region).

[0032] Figures 5A-5H Chronic recordings from the mTBI group of mice show altered power across different ECoG frequency bands. Figure 5A Representative 10-minute spectrograms are shown for sham-operated mice (left) and mTBI mice (right) at the same time point within the first 24 hours of mTBI, overlaid with ECoG traces from ipsilateral S1. The mTBI group spectrogram shows electrically recorded seizures, while the sham-operated spectrogram shows normal ECoG activity. The color bar represents power (mV2 / Hz). Figure 5B Representative seven-day spectrograms from sham-operated mice (left) and mice with mTBI (right) are shown, showing the power of different frequency bands two to three weeks after mTBI. Power bands were sampled every 30 minutes. Color bars represent power (mV2 / Hz). Figure 5C 、 5E , and 5G showed that in the first week after mTBI ( Figure 5C ), Week 3( Figure 5E ) and Week 11 ( Figure 5G ) Average power spectral density of ECoG activity in sham and mTBI mice. The inset in C shows examples of power spectral density plots from representative sham and mTBI mice. See Methods for details. Figure 5D 、 5F and 5H show the first week after mTBI ( Figure 5D ), Week 3( Figure 5F ) and Week 11 ( Figure 5H ) Two-way ANOVA of the mean power in each frequency band. Each point represents the power of one mouse. Data are representative of all mice recorded, even if they died or had battery failure before the experimental endpoint, using two-way ANOVA (*p < 0.05, **p < 0.01). n = seven sham-operated mice, 11 mTBI mice. One mouse died within two days after mTBI. The remaining mice were recorded during the first week after mTBI and then every other week until the eleventh week after mTBI. δ = 1-4 Hz, θ = 5-8 Hz, α = 9-12 Hz, σ = 13-15 Hz, β = 16-30 Hz, γ = 31-50 Hz.

[0033] Figures 6A-6E showed that anti-C1q antibodies had a modest effect on ECoG spectral features in mice with mTBI. Figure 6A Example spectrograms (top) and histograms (bottom) are shown for control-treated mice (left) and antibody-treated mice (right), showing power in different frequency bands one month after mTBI. Power bands were sampled every 30 minutes. The color bar represents power (mV2 / Hz). Figure 6B and 6D The first week after mTBI ( Figure 6B ) or the third week ( Figure 6D ) Average power spectral density of ECoG activity for control-treated and antibody-treated mTBI groups. Inset shows examples of power spectral density plots from representative control-treated and antibody-treated mTBI mice. Figure 6C and 6E The first week after mTBI ( Figure 6C ) and the third week ( Figure 6E ) Two-way ANOVA of mean power in each frequency band. Each point represents the power of one mouse. Data are representative of all mice recorded, even if they died before the end of treatment, using two-way ANOVA. n = seven control-treated mice, seven antibody-treated mice. δ = 1-4 Hz, θ = 5-8 Hz, α = 9-12 Hz, σ = 13-15 Hz, β = 16-30 Hz, γ = 31-50 Hz.

[0034] Figures 7A-7I Postmortem brain tissue from TBI patients was shown to display chronic inflammation eight days after TBI. Figure 7AShown is postmortem brain tissue from a control patient stained for HLA-DR, a marker for the MHC class II cell-surface receptor expressed on microglia and macrophages. Case information: Male, 78 years old. Scale bar, 1 cm. Figure 7B Shown is postmortem HLA-DR staining of brain tissue from a patient with TBI. Case information: 79-year-old male; fall accident; injury severity (GCS): moderate; CT scan: cerebral edema; no seizures (eight days after TBI); no history of neurological disease or evidence of cognitive decline based on the most recent clinical evaluation; no evidence of primary neurodegeneration or trauma-induced diffuse axonal injury. Scale bar, 1 cm. Figure 7C Display with ( Figure 7A ) Same as ( ), but stained for GFAP. Scale bar, 1 cm. Figure 7D Display with ( Figure 7B ) Same as ( ), but stained for GFAP. Scale bar, 1 cm. Figure 7E Display with ( Figure 7A ) Same as above, but stained for C1q. Scale bar, 1 cm. Figures 7F-7I Display with ( Figure 7B ) but stained for C1q. Scale bar, 1 cm ( Figures 7F-7G ) and 40μm( Figures 7H-7I ).

[0035] Figures 8A-8C showed that three weeks after mTBI, single-nucleus RNA sequencing did not reveal major differences in expression between sham and mTBI thalamic tissue. Figure 8A Shows the definition of Figure 3B Violin plots of key lineage genes for each cluster. Figure 8B Shown are UMAP images of sham and mTBI cell nuclei, colored by replicates (rep 1: n = five sham-operated mice, n = six mTBI mice, rep 2: n = four sham-operated mice, n = four mTBI mice). Figure 8C The percentage of nuclei collected from thalamic tissue from sham (n=4,908) and mTBI (n=4,338) thalamus within each lineage as defined in A) is shown. Recovery for each lineage was similar between replicates. Each dot represents one biological replicate.

[0036] Figures 9A-9H showed that the major complement markers found in thalamic tissue three weeks after mTBI were C1qa and C4b. Figure 9A Shown are the expression levels of Apoe and Cst3 in microglia and Apoe and Clu in astrocytes in sham and mTBI, analyzed by Wilcoxon rank sum test (adjusted p values ​​are shown above each violin plot). Figure 9B Violin plots of complement system components in the major lineages of delineated cells from sham and mTBI thalamic tissue are shown. Figure 9C Subclusters of the oligodendrocyte lineage are shown. Figure 9D Shows the same as ( Figure 9C ) in the same UMAP, expressed via C4b and colored with padding. Figure 9E Violin plots showing C4b expression in oligodendrocyte subclusters from sham-operated and mTBI mice analyzed using the Wilcoxon rank-sum test. C4b is differentially expressed in oligodendrocyte cluster 3 (Oligo 3). Analysis combined two technical replicates, representing nine sham-operated mice and ten mTBI mice. Each dot represents a single nucleus. Figure 9F Shows the same as ( Figure 9C ) are stained by the expression of Kirrel3 and Opalin, which are associated with mature oligodendrocytes; Enpp6, which is associated with differentiating oligodendrocytes; and the alarmin Il33, which is associated with oligodendrocyte maturation. Figure 9G RT-qPCR of C2 expression in RNA extracted from bulk cytoplasmic RNA is shown. Each dot represents bulk RNA extracted from one replicate (n = two biobanks, each dot represents n = three technical replicates). The first replicate included five sham-operated mice and six mTBI mice, and the second replicate included four sham-operated mice and four mTBI mice. Figure 9H Shown is a table summarizing the expression of all complement proteins from nuclear RNA-seq data and cytoplasmic RNA qPCR.

[0037] Figures 10A-10G described that single-nucleus RNA sequencing revealed an expression gradient within thalamic GABAergic neurons, but little difference between sham-operated and mTBI mice. Figure 10A UMAPs of all depicted nuclei (sham and mTBI) are shown, colored by Slc17a6 (left panel) and Slc17a7 (right panel) expression, using fill rendering. Nuclei within dashed circles were selected to subcluster GABAergic neurons. Figure 10B Shown are UMAP projections of GABAergic neurons from A) colored by novel subclusters. Figure 10C The percentage of each subcluster among the total number of GABAergic neuronal nuclei in nine sham-operated mice and ten mice with mTBI is shown. Each point represents one of two biological replicates (rep 1: n = five sham-operated mice, n = six mTBI mice, rep 2: n = four sham-operated mice, n = four mTBI mice). Figure 10DShown (top) is a UMAP projection colored by Ecel1 and Spp1 expression, with fill coloring. The "overlap" plot combines the two groups on the left, showing strong overlap between the nuclei of the two genes. (bottom) Same as the top plot, colored by Sst and Pvalb expression. Gray circles represent nuclei without expression. Figure 10E Shows ( Figure 10B ) Violin plots of Slc17a7, Gad2, Pvalb, and Sst from subclusters. Figure 10F A heat map of several genes related to neuronal function in GABAergic neuron subclusters is shown. The major categories of each gene are marked on the right. The colors represent proportional expression, normalized to the average value of all subclusters. The subclusters differ in the expression of adhesion and guidance molecules, which are related to axon guidance and growth. The adhesion and guidance molecules include genes in the cadherin (Cdh12, Cdh13, Cdh18) and protocadherin families, brain signal protein genes (Semaphorin genes (Sema5b, Sema3e) and ephrin receptor family genes (Epha5, Epha6). The subclusters also differ in the expression of glutamate receptors (Gria1, Grin2a, Grik4), extracellular matrix proteins (Col12a1, Col25a1) and cell signaling molecules (such as Nrg1, a member of the epidermal growth factor family that is believed to regulate Pvalb-positive neurons). Figure 10G Volcano plot showing differentially expressed genes across all GABAergic neurons between sham-operated and mTBI mice. Gray line indicates significance cutoff. Selected mitochondrial genes are labeled.

[0038] Figures 11A-11B showed that four months after mTBI, the damaged cortex and functionally connected thalamus showed chronic inflammation and neuronal loss. Figure 11A Close-up images of S1 (top), VB, and nRT (middle) stained for C1q, NeuN, GFAP, and IBA1 are shown, along with a confocal image of nRT (bottom). The site of injury in the right S1 cortex is marked with an asterisk. Arrows in nRT indicate the location of the confocal images. Scale bars, 300 μm (top / middle) and 20 μm (bottom). Figure 11B Quantification of the fluorescence ratio between the ipsilateral and contralateral regions in sham-operated and TBI mice is shown. Data represent all points from minimum to maximum, using the Mann-Whitney test and α = 0.05 (*p < 0.05, **p < 0.01). Analysis included four to six mice per group (n = one to three sections per mouse, one image per region).

[0039] Figures 12A-12C Depicts that three weeks after mTBI, C1q- / - mice display reduced inflammation and neuronal loss. Figure 12A Representative coronal brain sections from TBI C1q- / - mice stained for GFAP, IBA1, and NeuN are shown. The injury site in the right S1 cortex is marked with an asterisk. Scale bar, 1 mm. Figure 12B Close-up images of S1 (upper panel), VB, and nRT (lower panel) stained for GFAP, IBA1, and NeuN are shown. The site of injury in the right S1 cortex is marked with an asterisk. Scale bar, 500 μm. Figure 12C Quantification of fluorescence ratios and nRT neuron counts between ipsilateral and contralateral regions in sham-operated and TBI C1q- / - mice is shown. Data represent all points from minimum to maximum, using the Mann-Whitney test and α = 0.05 (*p < 0.05, **p < 0.01). Analysis included four to six mice per group (n = one to three sections per mouse, one image per region).

[0040] Figures 13A-13D Plasma and brain PK / PD are shown, demonstrating the presence of free drug and reduced C1q in sham-operated and TBI mice treated with anti-C1q drugs. Figure 13A Shown are the plasma levels of free drug, C1q-free and C1q-total measured using sandwich ELISA after treatment of TBI groups and sham-operated mice with two doses of 100 mg / kg anti-C1q or isotype control antibody. The dashed line shows the lower limit of quantitation. Figures 13B-13D Shown are free drug ( Figure 13B )、C1q-free ( Figure 13C ) and C1q-total ( Figure 13D ) levels. Untreated mice served as negative controls. The dashed line indicates the lower limit of quantification. Data represent all points from minimum to maximum, using the Mann-Whitney test with α = 0.05 between TBI control and TBI anti-C1q (*p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001). Analysis included three to 15 mice per group.

[0041] Figures 14A-14D showed that mice with mTBI had spontaneous epileptiform events in the theta to alpha frequency range that were time-locked to thalamic bursting four weeks after mTBI. Figure 14A Schematic diagrams showing the recording sites for the in vivo experiments. Left: ECoG recording sites and mTBI locations are shown on the mouse skull. Right: Approximate locations of tungsten depth electrodes implanted unilaterally for nRT. Figure 14B Shown are representative ECoG traces from cortical recording sites and multiunit traces from nRT demonstrating spontaneous epileptiform events. Figure 14CShown is a power spectrum analysis showing the average power across different frequency bands during the first 15 min of baseline ECoG signals from the ipsilateral S1 cortex of sham-operated and TBI mice. Figure 14D Shown are periodograms showing power across frequencies obtained from the first 15 minutes of baseline ECoG signals from the ipsilateral S1 cortex of representative sham-operated and TBI mice. Data represent mean ± SEM and were analyzed using the Mann-Whitney test with α = 0.05 (*p < 0.05, **p < 0.01). 12 to 14 mice were included in the analysis per group.

[0042] Figures 15A-15D Anti-C1q antibodies were shown to have a chronic disease-modifying effect on ECoG power in mice with mTBI. Figure 15A Example spectrograms (top) and histograms (bottom) are shown for control-treated mice (left) and antibody-treated mice (right), showing power in different frequency bands 2.5 months after TBI (i.e., four weeks after the end of treatment). Power bands were sampled every 30 minutes. Figure 15B Cumulative distribution functions of control-treated and antibody-treated cohorts sampled at different frequency bands on the first day after TBI are shown. We sampled 48 points within the first 24 hours of each recording. Figure 15C Shown is the same as in B, but three weeks after TBI. We sampled 232 points between 15.25 and 20.1 days after the start of each recording. Figure 15D Shown are the same as in B, but from 9 to 15 weeks after TBI. We sampled 296 points between 104.6 and 110 days after the start of each recording. Data represent all mice recorded, even if they died before the end of treatment. One control-treated mouse and one antibody-treated mouse died within three weeks after TBI, two control-treated mice died within six weeks after TBI, and the remaining mice were recorded for at least nine weeks after TBI. At 24 hours, n = seven control-treated mice, seven antibody-treated mice. At three weeks, n = seven control-treated mice, seven antibody-treated mice. At 9 to 15 weeks, n = seven control-treated mice, four antibody-treated mice. δ = 1–4 Hz, θ = 5–8 Hz, α = 9–12 Hz, σ = 13–15 Hz, β = 16–30 Hz, γ = 31–50 Hz. ns = p > 0.05.

[0043] Figures 16A-16D showed that anti-C1q antibodies restored reduced sleep spindles three weeks after mTBI. Figure 16A Representative ECoG recordings from sham-operated and mTBI mice three weeks after mTBI are shown. Traces represent bandpass (BP) filtered ECoGs. Horizontal lines show detected spindle waves. Arrows indicate epileptic spikes. Figure 16B Shows the Figure 16A The same mTBI mice were treated with isotype control or anti-C1q antibody. Figure 16C The ratio of sleep spindles in the ipsilateral ECoG to the contralateral ECoG detected within a 12-hour window is shown. Data represent mean ± SEM and were analyzed using the Mann-Whitney rank sum test with α = 0.05 (*p < 0.05, **p < 0.01). Analyses included n = six sham-operated mice, n = nine mTBI mice (left); n = seven control-treated mTBI mice, and n = seven antibody-treated mTBI mice (right). Figure 16D Shows ( Figure 16C ) in the contralateral and ipsilateral ECoG of mice. Data represent the mean ± SEM analyzed using a one-way Kruskal-Wallis rank variance analysis, all pairwise multiple comparison procedures (Holm-Sidak method), α = 0.05 (*p < 0.05, **p < 0.01). Gray lines represent the contralateral and ipsilateral data for each mouse.

[0044] Figures 17A-17E showed that anti-C1q antibodies reduced focal epileptic spikes that appeared three weeks after mTBI. Figure 17A Representative ECoG recordings from sham-operated and mTBI mice three weeks after mTBI are shown. The horizontal dashed line indicates the spike detection threshold. The vertical red line indicates detected spikes. Figure 17B A shows the Figure 17A The same mTBI mice were treated with isotype control or anti-C1q antibody. Traces AB are from the onset of NREM sleep. Figure 17C The number of epileptic spikes detected within a 12-hour window is shown. Data represent mean ± SEM and were analyzed using the Mann-Whitney rank sum test (*p < 0.05, **p < 0.01). Inset: Average epileptic spikes from mTBI mice are shown in (B) (n = 592 spikes; mean (black) ± SD (grey). Analysis included n = six sham-operated mice, n = nine mTBI mice (left); n = seven control-treated mTBI mice, and n = six antibody-treated mTBI mice. Figures 17D-17E Shows the Figure 17C The ratio of the number of epileptic spikes to sleep spindles in mice in Figure 2. Individual points represent each mouse, and error bars represent the mean ± SEM on both axes.

[0045] Figure 18Sleep spindle detection is shown. Simultaneously recorded ECoG signals from the cortex surrounding the mTBI and the contralateral cortex (black) were bandpass filtered 8-15 Hz (grey trace) and a sleep spindle detection threshold (Thr.) was applied. The depicted recordings are from anti-C1q treated mTBI mice three weeks after mTBI (with Figure 17B same as for mice in the study). DETAILED DESCRIPTION

[0046] This detailed description should not be considered as limiting, but is only for the purpose of illustrating the general principles of the present invention. The section titles and overall structure of this detailed description are for convenience only and are not intended to limit the present invention.

[0047] It is estimated that in a lifetime (80 years old), the probability of suffering from epilepsy is about 3%. In about 30% of cases, there is identifiable brain damage that triggers epilepsy (symptomatic epilepsy). Another 30% of patients are speculated to have symptomatic epilepsy, and their cause of disease has not yet been identified. Brain damage, such as traumatic brain injury (TBI), stroke, status epilepticus and infection / inflammation are some causes of acquired epilepsy, which usually occurs after the incubation period and is often progressive (that is, over time, seizures become more frequent and severe). Epileptic seizures may also occur in patients in the recovery period after brain surgery. In addition, for about 1% of the crowd, epileptic seizures are spontaneous, without any obvious reason or any other neurological abnormality. This type of spontaneous epilepsy is called idiopathic epilepsy, and is considered to be hereditary.

[0048] Epilepsy is not a specific disease, or even a single syndrome, but rather a broad group of conditions arising from a number of disorders of brain function, which may be secondary to a variety of pathological processes. The terms convulsive disorder, paroxysmal disorder, and seizure are used synonymously for epilepsy as they all refer to recurrent paroxysmal attacks of brain dysfunction manifested by stereotyped behavioral changes. An epileptic seizure is considered to be an abrupt change in behavior, the result of hypersynchronous firing of neuronal networks involving the cortex. An epileptic seizure may also be a natural response of the normal brain to a brief disturbance in function and is not necessarily an indication of an epileptic disorder. Such seizures are often referred to as provocative, acute symptomatic, or reactive.

[0049] Several genes encoding protein subunits of voltage-gated and ligand-gated ion channels are associated with various forms of epilepsy and infantile epilepsy syndromes. Patients with uncontrolled seizures suffer significant morbidity and mortality.

[0050] In epilepsy, the brain undergoes permanent pathophysiological or structural changes, leading to abnormal, hypersynchronous neuronal firing. Repeated seizures can lead to progressive brain damage. For example, a gradual decrease in hippocampal volume over time has been reported as a function of seizure frequency. Several clinical and experimental data suggest that failure of the blood-brain barrier (BBB) ​​can trigger chronic or acute seizures.

[0051] More than forty types of epileptic seizures have been characterized, and these seizures are divided into generalized (seizures in both hemispheres of the brain) and partial (focal; seizures in one part of the brain). Generalized seizures are further divided into absence seizures, myoclonic seizures, atonic seizures, and tonic seizures, while partial seizures are further divided into simple seizures and complex seizures. Partial seizures account for about sixty percent of all adult cases, and temporal lobe epilepsy (TLE) is the most common form of partial seizures. TLE patients usually have a history of early risk factors, such as febrile seizures, status epilepticus, and infection. Before uncontrolled partial seizures begin, there may be a period of seizure-free period. There are some progressive features, such as increased frequency of seizures and decreased cognitive ability.

[0052] The cause of epilepsy remains a mystery. However, there is evidence that activation of immune pathways plays a role in human epilepsy, and that this inflammatory response contributes to the generation and recurrence of seizures and the neuronal damage associated with them. Astrocytes are known to contribute to the inflammatory environment of the central nervous system by producing a wide range of immune-related molecules. They can express class II major histocompatibility association antigens and produce a variety of chemokines and cytokines. These immune factors may also activate microglia.

[0053] For example, in patients with temporal lobe epilepsy (TLE), there is evidence of microglial activation within the hippocampus, providing evidence for an activated immune response. Nuclear factor-κB is overexpressed in reactive astrocytes and surviving neurons in human hippocampal sclerosis specimens. Furthermore, there is significant and sustained activation of the IL-1β system, involving both activated glial and neuronal cells. In contrast, only a few adaptive immune cells (CD3 / CD8-positive T lymphocytes) have been detected in human mesial TLE specimens. Furthermore, gene expression profiling supports the activation of inflammatory pathways in human TLE. Recent studies have demonstrated differential correlations between the expression of key inflammatory factors and seizure frequency in patients with drug-resistant mesial TLE. Toll-like receptor 4 (TLR4) gene expression—previously shown in animal models of TLE to be a key trigger of inflammation that induces the transcription of several cytokines—is directly correlated with seizure frequency, while expression of activating transcription factor 3 (a negative regulator of TLR4) and IL-8 is inversely correlated with seizure frequency.

[0054] The interaction between dysregulated, persistent inflammation, blood-brain barrier damage, and uncontrolled seizures creates a self-perpetuating cycle, leading to uncontrolled inflammation that triggers the progression of various epileptic conditions, including TLE. For example, a key inflammatory mediator is the complement system. The complement system is a protein cascade involved in the immune response, composed of approximately 30 fluid-phase, cell membrane-associated proteins. Activated products of the cascade contribute to the production of other inflammatory mediators, thereby promoting tissue damage at the site of inflammation. Although synthesis of complement system components occurs primarily in the liver, under pathological conditions, both glial cells and neurons express these inflammatory mediators. C3a and C5a are the most potent proinflammatory molecules produced in response to complement activation. C1q, the initiator of the classical complement cascade, recognizes neuronal synapses under stress. Activation of C1q leads to the deposition of downstream complement components C4b and C3b on the synaptic surface, leading to recognition by immune cells and physical elimination of the synapse. Activation of the complement system also results in the formation of the membrane attack complex, which damages or lyses target cells by forming pores in the phospholipid bilayer. Neurons are particularly sensitive to complement-mediated damage. In addition, although complement factors may invade the brain via a leaky blood-brain barrier, some increased expression may originate from activated glial cells. Interestingly, sequential infusion of individual proteins of the membrane attack pathway (C5b6, C7, C8, and C9) into the hippocampus of awake, freely moving rats induced behavioral and electrical seizures as well as cytotoxicity, suggesting a role for the complement system in epileptogenesis.

[0055] Furthermore, traumatic brain injury (TBI) affects approximately 69 million people worldwide each year and can lead to cognitive impairment, sensory processing difficulties, sleep disruption, and, as mentioned above, the development of epilepsy. Most of these adverse health consequences emerge months or years after TBI and are caused by indirect secondary injuries that lead to long-term consequences of the initial impact.

[0056] Although TBI severely damages the cerebral cortex, most TBI-related disabilities reflect secondary injury that develops over time. The thalamus is a likely site of secondary damage because of its interconnectedness with the cerebral cortex. Using a mouse model of mild cortical injury (mTBI) that does not directly damage subcortical structures, we found chronic increases in C1q expression, particularly in the corticothalamic circuit. Increased C1q expression colocalized with neuronal loss and chronic inflammation and was associated with disruption of sleep spindles and the emergence of epileptic activity. Blockade of C1q abolished most of these effects, implicating C1q as a disease modifier in mTBI. Single-nucleus RNA sequencing demonstrated that microglia are the source of thalamic C1q, which likely acts on subsets of oligodendrocytes and astrocytes. The cortex is often the primary site of injury because it lies directly beneath the skull and is a component of many larger circuits, including the cortical-thalamic-cortical loop. This circuit is important for sensory processing, attention, cognition, and sleep, all of which may be impaired by TBI. The thalamus itself, while not acutely injured in TBI, undergoes secondary damage, likely due to its long-range interconnectedness with the cerebral cortex. Structural changes in the thalamus have been linked to many long-term TBI-related health outcomes, including fatigue and cognitive impairment, and TBI patients exhibit secondary and chronic neurodegeneration and inflammation in thalamic nuclei.

[0057] Chronic neuroinflammation is a common feature of secondary injury sites. However, most attempts to improve cognitive outcomes after TBI with broad-spectrum anti-inflammatory agents have failed, probably because there are many inflammatory pathways that play both protective and pathogenic roles at different times. A potential mediator of inflammation and injury after TBI is the complement pathway, which is activated in the peri-injury area of ​​brain lesions in humans and rodents. Complement activation leads to inflammation and neurotoxicity in central nervous system injuries and is increased in the human brain with injury, epilepsy and Alzheimer's disease. Abnormal activation of C1q, the initiator molecule of the classical complement cascade, can trigger the elimination of functional synapses and lead to the progression of neurodegenerative diseases. On the other hand, C1q is involved in normal synaptic pruning during development, and the complement system plays an important role in brain homeostasis by clearing cellular debris and protecting the central nervous system from infection.

[0058] This article provides insights into the role of the C1q pathway in secondary injury to corticothalamic circuits after TBI in a mechanically tractable and highly reproducible mouse model of mild cortical injury. This model identified factors such as the therapeutic window, inflammatory phenotype, and extent of secondary injury that support targeted approaches in the treatment of post-TBI outcomes.

[0059] A powerful tool we have used to study the entire somatosensory corticothalamic circuitry after TBI is chronic ECoG recordings, specifically to investigate the progression of post-traumatic epileptogenesis and changes in cortical rhythms up to four months after TBI. Using this electrophysiological approach at the cellular and circuit levels, we show that TBI alters the synaptic properties of neurons in the nucleus reticularis thalamus (nRT) and is associated with increased C1q accumulation, which may mediate pathological states in the corticothalamic circuitry, including increased broadband activity.

[0060] nRT is a site of long-term secondary injury after TBI

[0061] Previous observations following severe head injury have shown neurodegeneration in human nRT (42). Our study demonstrates that even mild cortical injury leads to neuronal loss in nRT three weeks after injury. This neurodegeneration may be due to loss of cortical input leading to excitotoxicity in nRT, a vulnerable brain region likely due to a high density of axonal afferents from the cortex (42). Our RNA sequencing results also identified increased expression of genes related to mitochondrial function in mTBI thalamic tissue across all GABAergic neuron subclusters. This observation points to mitochondria-mediated cell death as another potential mechanism of post-TBI neurodegeneration.

[0062] The loss of neurons in the nRT could explain some of the synaptic changes in this region. In particular, the frequency of IPSCs in nRT neurons was reduced three weeks after TBI. In many microcircuits, reduced inhibition of GABAergic neurons results in a net increase in inhibition. In contrast, the loss of GABAergic inhibition in the nRT leads to hyperexcitability in corticothalamic circuits and can even trigger epileptiform activity. Indeed, GABAergic connections within the nRT are important for coordinating the inhibitory output of excitatory thalamic nuclei and controlling oscillatory thalamic activity, and their loss is detrimental to corticothalamic circuits. The death of GABAergic neurons within the nRT may contribute to the reduction of inhibition within the nRT. This reduced inhibition may lead to a loss of feedforward GABAergic inhibition, which may lead to an increased susceptibility to seizures and an increased likelihood of developing post-traumatic epileptic activity.

[0063] Deficits were also observed in nRT EPSCs, specifically lower frequency and amplitude and slower kinetics. These changes resemble findings in a mouse model of epilepsy lacking GluA4 AMPA receptors at cortico-nRT glutamatergic synapses. This defect leads to loss of feedforward inhibition in the thalamus and epileptic activity. Thus, alterations in nRT EPSCs appear to contribute to hypersynchrony in corticothalamic circuits and seizures, but may be due to loss of cortical glutamatergic input to nRT after TBI.

[0064] Given that the changes we found in the corticothalamic circuit, particularly in nRT, are associated with epileptic activity and cognitive deficits, these results identify this circuit as a novel potential target for treating long-term TBI outcomes. Of particular interest is the important role that sleep spindles play in cognitive function. Our findings identify C1q as a target for treating sleep spindles and preventing epileptic spikes after mTBI.

[0065] Unlike nRT neurons, cortical neurons, such as layer 5 pyramidal neurons and GABAergic fast-firing interneurons, were not altered by mild TBI (mTBI) at chronic time points. These observations suggest the existence of homeostatic mechanisms that restore or reduce chronic hyperexcitability after cortical TBI. They also confirm that at least some long-term consequences of TBI are undoubtedly caused by nRT dysfunction, not solely by cortical damage. In this regard, it is interesting to see that although cortical neurons appear to have normal excitability and synaptic function at chronic stages, ECoG reveals “focal” deficits in sleep spindles and epileptic spikes. This observation is consistent with previous magnetoencephalography studies in humans with mild TBI (mTBI), EEG studies in humans with severe TBI, and EEG studies in rats with severe TBI, which have observed increased delta activity at early time points after TBI. Under normal circumstances, delta activity is associated with slow-wave sleep, quiet wakefulness, and higher-order cognitive functions. In the context of injury, delta waves are associated with white matter lesions. Therefore, the major long-term effects of mild TBI (mTBI) are at the thalamic end of the cortico-thalamo-cortical loop. Given the emerging role of nRT in generating localized sleep spindles in sensory cortex, secondary damage to nRT may be at least partially responsible for the “localized” loss of sleep spindles in the cortex.

[0066] Chronic C1q as a disease modifier

[0067] C1q has a well-documented role in normal brain function, such as synaptic pruning during development, and is involved in several neurological disorders, including severe TBI. Furthermore, C1q expression is highly increased in corticothalamic circuits up to four months after TBI. The studies disclosed here focus on corticothalamic circuits and, using electrophysiological recordings, provide the first functional characterization of corticothalamic circuits after mTBI and demonstrate for the first time that the loss of sleep spindles and the development of epileptic spikes after mTBI involve the C1q pathway.

[0068] Although mice with mild TBI (mTBI) do not develop chronic GTCS to determine whether blocking C1q has an anti-seizure effect, many other protective effects of anti-C1q antibodies have been observed, including reduced inflammation and neurodegeneration, and restoration of altered cortical states after TBI, such as protection against disruption of sleep spindles and epileptic spikes. Based on these observations and previous literature suggesting differences between protective and deleterious inflammatory cell types, C1q plays distinct roles at different stages of pathology. At the time of injury, C1q contributes to the formation of glial scars, thereby limiting the extent of damage to the primary site of cortical injury. However, in the chronic stage, increased C1q promotes chronic inflammation and secondary neurodegeneration in nRT.

[0069] The cortex also exhibited an increase in C1q, but it did not appear to have a damaging effect at that site or time point, or it may have counteracted the initial protective effect, because, unlike the thalamus, neuronal physiology in the cortex of sham-operated and TBI mice was similar at chronic time points. These findings suggest a time window in which anti-C1q treatment could prevent secondary damage to the thalamus without compromising homeostatic recovery in the cortex.

[0070] Our RNA sequencing results indicate that microglia are the primary source of C1q, as well as C4b, for astrocytes and oligodendrocytes, a finding that may point to additional cell-specific therapeutic targets both upstream (microglia) and downstream (astroglia and oligodendrocytes) of the C1q molecule itself. C4 also appears to mediate injury after severe TBI, as shown by the reduced motor deficits in C4- / - mice. The lack of Hc expression in our sequencing data suggests that the mechanism of nRT neuronal death is not membrane attack complex-mediated lysis, although this mechanism cannot be determined by sequencing alone.

[0071] The studies disclosed herein identify C1q as a disease modifier that can be targeted within a time window (in this study, treatment started 24 hours after injury) to treat the devastating consequences of TBI. Thalamic C1q can also serve as a biomarker to help identify individuals who are likely to develop long-term secondary injuries. This study is the first to perform electrophysiological recordings from the cortex and thalamus at chronic time points after mTBI and identify neuronal death and reduced IPSCs in nRT as chronic consequences of cortical injury. Furthermore, by showing the physiological chronic consequences of mTBI in nRT, we identify nRT as a novel target for treating post-TBI consequences such as altered sensory processing, sleep disruption, and epilepsy.

[0072] Although these studies have implicated the role of different immune pathways, TLE, and secondary injury following TBI (e.g., TBI-induced epilepsy), the art needs new compositions and methods for preventing and treating epilepsy and its associated progression. Therefore, inhibiting early complement activation pathways may be a promising strategy for treating epilepsy, for example, using anti-C1q, anti-C1r, and anti-C1s antibodies that inhibit early complement activation (including complement activation pathways). The antibodies can be monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and / or antibody derivatives.

[0073] Neutralizing the activity of complement factors such as C1q, C1r, or C1s inhibits classical complement activity and slows or prevents epilepsy, such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy. Disclosed herein are methods related to neutralizing complement factors (e.g., C1q, C1r, or C1s) in epilepsy, such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy.

[0074] All sequences mentioned in this disclosure are incorporated by reference into U.S. Patent No. 10,316,081, U.S. Patent Application No. 14 / 890,811, U.S. Patent No. 8,877,197, U.S. Patent No. 9,708,394, U.S. Patent Application No. 15 / 360,549, U.S. Patent No. 9,562,106, U.S. Patent No. 10,450,382, U.S. Patent No. 10,457,745, International Patent Application No. PCT / US2018 / 022462, each of which disclosed antibodies and related compositions are hereby incorporated by reference.

[0075] In certain aspects, disclosed herein are methods of preventing, reducing the risk of developing epilepsy, or treating epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy) comprising administering to a subject an inhibitor of a complement pathway.

[0076] Disclosed herein is a method for inhibiting epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy) comprising administering an antibody (such as an anti-Clq antibody, an anti-Clr antibody, or an anti-Cls antibody) to a patient. In certain preferred embodiments, the antibody binds to Clq, Clr, or Cls and inhibits complement activation. The antibody can be a monoclonal antibody, a chimeric antibody, a humanized antibody, an antibody fragment thereof, and / or an antibody derivative thereof.

[0077] Full-length antibodies can be prepared by using recombinant DNA engineering techniques. Such engineered versions include versions produced by, for example, inserting, lacking or changing the natural antibody variable region in the amino acid sequence of a natural antibody. The specific examples of this type include those engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from a kind of antibody and from the remainder of the variable region domain of a second antibody. The DNA encoding the antibody can be prepared by deleting all parts except the desired part in the DNA encoding the full-length antibody. The DNA encoding the chimeric antibody can be prepared by recombining the DNA of the variable region of the variable region sequence of the mammalian variable region substantially or specifically encoding human constant region and coding substantially or specifically deriving from the human. The DNA encoding the humanized antibody can be prepared by recombining the DNA encoding the constant region and the variable region except the complementary determining region (CDR) of the corresponding human antibody district and coding substantially or specifically deriving from the human CDR of the mammalian variable region.

[0078] Suitable sources of DNA molecules encoding antibodies include cells expressing full-length antibodies, such as hybridomas. For example, antibodies can be isolated from host cells expressing expression vectors encoding the heavy and / or light chains of the antibody.

[0079] Antibody fragments and / or antibody derivatives can also be prepared using recombinant DNA engineering techniques, including manipulation and re-expression of DNA encoding the variable and constant regions of antibodies. Standard molecular biology techniques can be used to modify, add or delete more amino acids or domains as needed. As used herein, the terms "variable" and "constant" regions still encompass any changes to the variable or constant regions. In some cases, by altering the C H A stop codon was introduced immediately after the codon for the interchain cysteine ​​of 1, and PCR was used to generate antibody fragments such that C H The 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.

[0080] The antibodies of the present disclosure can be derived from any antibody isotype ("class"), including, for example, IgG, IgM, IgA, IgD, and IgE, and subclasses thereof, including, for example, IgG1, IgG2, IgG3, and IgG4. In certain preferred embodiments, the heavy and light chains of the antibodies are from IgG. The heavy and / or light chains of the antibodies can be from mouse IgG or human IgG. In certain other preferred embodiments, the heavy and / or light chains of the antibodies are from human IgG1. In yet other preferred embodiments, the heavy and / or light chains of the antibodies are from human IgG4.

[0081] In some embodiments, the inhibitor is an antibody, such as an anti-Clq antibody, an anti-Clr antibody, or an anti-Cls antibody. The anti-Clq antibody can inhibit the interaction between Clq and an autoantibody, or between Clq and Clr, or between Clq and Cls. The anti-C1r antibody can inhibit the interaction between Clr and Clq, or between Clr and Cls. The anti-C1r antibody can inhibit the catalytic activity of Clr, or the anti-C1r antibody can inhibit the processing of pro-Clr into an active protease. The anti-C1s antibody can inhibit the interaction between Cls and Clq, or between Cls and Clr, or between Cls and C2 or C4, or the anti-C1s antibody can inhibit the catalytic activity of Cls, or it can inhibit the processing of pro-Cls into an active protease. In some cases, the anti-Clq, anti-C1r, or anti-C1s antibody promotes the clearance of Clq, Clr, or Cls from the circulation or tissues.

[0082] The antibodies disclosed herein can be, for example, monoclonal antibodies that bind to mammalian C1q, C1r, or C1s, preferably human C1q, C1r, or C1s. The antibodies can be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, antibody fragments, or antibody derivatives thereof. The antibodies disclosed herein can also cross the blood-brain barrier (BBB). The antibodies can activate BBB receptor-mediated transport systems, such as systems that utilize the insulin receptor, transferrin receptor, leptin receptor, LDL receptor, or IGF receptor. The antibodies can be chimeric antibodies with sufficient human sequences that are suitable for administration to humans. The antibodies can be glycosylated or non-glycosylated; in some embodiments, the antibodies are glycosylated, for example, with a glycosylation pattern produced by post-translational modification in CHO cells. In some embodiments, the antibodies are produced in Escherichia coli (E coli).

[0083] The antibody can be a bispecific antibody that recognizes a first and a second antigen, for example, the first antigen is selected from C1q, C1r and C1s, and / or the second antigen is an antigen that allows the antibody to cross the blood-brain barrier, such as selected from transferrin receptor (TR), insulin receptor (HIR), insulin growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, llama single domain antibody, TMEM 30 (A), protein transduction domain, TAT, Syn-B, penetratin, polyarginine peptide, angiopoietin peptide or ANG1005.

[0084] The antibodies disclosed herein can bind to and inhibit the biological activity of C1q, C1r, C1s, or C1. For example, (1) binding of C1q to an autoantibody, (2) binding of C1q to C1r, (3) binding of C1q to C1s, (4) binding of C1q to phosphatidylserine, (5) binding of C1q to pentraxin-3, (6) binding of C1q to C-reactive protein (CRP), (7) binding of C1q to globular C1q receptor (gC1qR), (8) binding of C1q to complement receptor 1 (CR1), (9) binding of C1q to beta-amyloid protein, or (10) binding of C1q to calreticulin. In other embodiments, another example of a biological activity of C1q 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) microglia activation, (10) Arthritis reaction, (11) phagocytosis of synapses or nerve terminals, and (12) activation of complement receptor 3 (CR3 / C3) expressing cells.

[0085] In some embodiments, CH50 hemolysis includes human, mouse, and / or rat CH50 hemolysis. In some embodiments, the antibody is capable of neutralizing at least about 50% to at least about 95% of CH50 hemolysis. The antibody is also capable of neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml, or less than 20 ng / ml.

[0086] Other in vitro assays for measuring complement activity include ELISA assays for measuring cleavage products of complement components or complexes formed during complement activation. Complement activation via the classical pathway can be measured by tracking the levels of C4d and C4 in serum. Activation of the alternative pathway can be measured in an ELISA by assessing the levels of circulating Bb or C3bBbP complexes. In vitro antibody-mediated complement activation assays can also be used to assess inhibition of C3a production.

[0087] The antibodies of the present disclosure may be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, antibody fragments thereof, or derivatives thereof.

[0088] The antibodies of the present disclosure may also be antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, or single-chain antibody molecules.

[0089] Disclosed herein are methods of administering a second agent, such as a second inhibitor, to a subject. In some embodiments, the second inhibitor can be an antibody (e.g., an anti-Clq antibody, an anti-Clr antibody, or an anti-Cls antibody). In other embodiments, the second inhibitor can be an inhibitor of antibody-dependent cellular cytotoxicity, an alternative complement activation pathway; and / or an inhibitor of the interaction between an autoantibody and an autoantigen.

[0090] In some embodiments, a method for determining the risk of a subject developing epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy) is provided, comprising: (a) administering an antibody (i.e., an anti-C1q, anti-C1r, or anti-C1s antibody) to the subject, wherein the antibody is conjugated to a detectable label; (b) detecting the detectable label to measure the amount or location of C1q, C1r, or C1s in the subject; and (c) comparing the amount or location of one or more of C1q, C1r, or C1s to a reference, wherein the risk of developing epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy) is characterized based on the comparison of the amount or location of one or more of C1q, C1r, or C1s to the reference. The detectable label can include a nucleic acid, an oligonucleotide, an enzyme, a radioisotope, biotin, or a fluorescent label. In some cases, the antibody can be labeled with a coenzyme (e.g., biotin) using a biotinylation process. When biotin is used as a label, detection of the antibody is accomplished by the addition of a protein (e.g., avidin or its bacterial counterpart, streptavidin), any of which can be conjugated to a detectable marker (e.g., the dyes described above), a fluorescent marker (e.g., fluorescein), a radioisotope, or an enzyme (e.g., peroxidase). In some embodiments, the antibody is an antibody fragment (e.g., a Fab, Fab'-SH, Fv, scFv, or F(ab')2 fragment).

[0091] The antibodies disclosed herein may also be coupled to a labeling group, such as a radioisotope, a radionuclide, an enzymatic group, a biotinyl group, a nucleic acid, an oligonucleotide, an enzyme, or a fluorescent label. The labeling group may be coupled to the antibody via a spacer arm of any suitable length to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used to prepare such labeled antibodies.

[0092] Various routes of administration are contemplated. Such methods of administration include, but are not limited to, topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intrathecal, intranasal, and intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. For the treatment of central nervous system conditions, the antibody may be suitable for crossing the blood-brain barrier via non-invasive peripheral administration routes, such as intravenous, intramuscular, subcutaneous, intraperitoneal, or even oral administration.

[0093] Suitable antibodies include antibodies that bind complement components C1q, C1r, or C1s. Such antibodies include monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and / or antibody derivatives thereof.

[0094] Preferred antibodies are monoclonal antibodies, which can be produced by immunizing rodents (e.g., mice, rats, hamsters, and guinea pigs) with (1) natural complement components (e.g., C1q, C1r, or C1s) derived from enzymatic digestion of purified complement components from human plasma or serum, or (2) recombinant complement components expressed by eukaryotic or prokaryotic systems, or fragments derived therefrom. Other animals can be used for immunization, such as nonhuman primates, transgenic mice expressing human immunoglobulins, and severe combined immunodeficiency (SCID) mice transplanted with human B lymphocytes.

[0095] Polyclonal and monoclonal antibodies are naturally produced as immunoglobulin (Ig) molecules by the immune system in response to pathogens. The approximately 150 kDa IgG1 molecule is the predominant form in human serum at a concentration of 8 mg / ml and is composed of two identical approximately 50-kDa heavy chains and two identical approximately 25-kDa light chains.

[0096] Hybridomas can be produced by fusing B lymphocytes from immunized animals with myeloma cells using conventional procedures. Furthermore, anti-C1q, anti-C1r, or anti-C1s antibodies can be produced by screening recombinant single-chain Fv or Fab libraries from human B lymphocytes in a phage display system. The specificity of MAbs for human C1q, C1r, or C1s can be tested by enzyme-linked immunosorbent assay (ELISA), Western immunoblotting, or other immunochemical techniques.

[0097] The antibodies identified during the screening process can be evaluated for their inhibitory activity against complement activation by hemolytic assays using unsensitized rabbit or guinea pig RBCs for the alternative complement pathway or sensitized chicken or sheep RBCs for the classical complement pathway. Hybridomas that exhibit specific inhibitory activity against the classical complement pathway are cloned by limiting dilution. Antibodies are purified by the above assays for characterization of their specificity for human C1q, C1r, or C1s.

[0098] Based on the molecular structure of the variable regions of anti-C1q, anti-C1r, or anti-C1s antibodies, molecular modeling and rational molecular design can be used to generate and screen small molecules that mimic the molecular structure of the antibody binding region and inhibit the activity of C1q, C1r, or C1s. These small molecules can be peptides, peptidomimetics, oligonucleotides, or organic compounds. Mimetic molecules can be used as inhibitors of complement activation in inflammatory indications and autoimmune diseases. Alternatively, large-scale screening procedures commonly used in the art can be used to isolate suitable small molecules from combinatorial compound libraries.

[0099] Suitable dosages for the antibodies disclosed herein may be between 10 and 500 μg / ml serum. The actual dosage can be determined in clinical trials following conventional methods for determining optimal dosages, i.e., administering various doses and determining which doses provide suitable efficacy without undesirable side effects.

[0100] Before the advent of recombinant DNA technology, proteolytic enzymes (proteases) that cleave polypeptide sequences were used to dissect the structure of antibody molecules and determine which parts of the molecule were responsible for their various functions. Limited digestion with papain cleaves the antibody molecule into three fragments. Two of the 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 consisting of a V chain that is identical to the heavy chain. H and C H The other fragments do not have antigen binding activity but were initially observed to readily crystallize and were therefore named Fc fragments (fragment crystallizable).

[0101] Fab molecules are artificial fragments of Ig molecules of approximately 50 kDa, whose heavy chains lack the constant domain C H 2 and C H 3. Two heterophiles (V L -V H and C L -C H 1) Domain interaction is the basis of the double-chain structure of Fab molecules, which is achieved through C L with C H Fab and IgG have the same antigen binding site formed by six complementary determining regions (CDRs), V L and V H Three of each (LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3). CDRs define the highly variable antigen-binding site of an antibody. The highest sequence variation is found in LCDR3 and HCDR3, which are respectively represented by V and V in the natural immune system. L and J L Gene or V H 、D H and J H The rearrangement of genes produces the LCDR3 and HCDR3, which usually form the core of the antigen-binding site. The conserved regions that connect and display the six CDRs are called framework regions. In the three-dimensional structure of the variable domain, the framework region forms a sandwich of two antiparallel β-sheets, which are connected by the outer hypervariable CDR loops and the inner conserved disulfide bonds.

[0102] definition

[0103] As used herein in the specification, "a" or "an" can mean one or more. As used herein in one or more claims, when used in conjunction with the word "comprising," the word "a" or "an" can mean one or more than one. For example, a reference to an "antibody" is a reference to one or more antibodies. As used herein, "another" can mean at least a second or more.

[0104] As used herein, administration "in conjunction" with another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a co-formulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.

[0105] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The term "antibody" is used herein in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, antibody fragments (as long as they exhibit the desired biological activity), and antibody derivatives.

[0106] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). H and V L Paired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0107] The L chains from any vertebrate species can be divided into two clearly distinct types, called κ ("kappa") and λ ("lambda"), based on the amino acid sequence of their constant domains. Immunoglobulins can be divided into different classes, or isotypes, depending 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, whose heavy chains are designated α ("alpha"), δ ("delta"), ε ("epsilon"), γ ("gamma"), and μ ("mu"), respectively. Based on relatively minor differences in CH sequence and function, the γ and α 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 different classes of immunoglobulins are well known and generally described, for example, in Abbas et al., Cellular and Molecular Immunology, 4th ed. (WB Saunders Co., 2000).

[0108] The term "agent" as used herein describes any molecule (e.g., protein or drug) that has the ability to modulate synaptic loss, particularly through the complement pathway. Candidate agents also include genetic elements (e.g., antisense and RNAi molecules that inhibit C1q expression) and constructs encoding complement inhibitors (e.g., CD 59). Candidate agents encompass many chemical categories, although they are typically organic molecules comprising small organic compounds with a molecular weight greater than 50 and less than about 2,500 daltons. Candidate agents contain functional groups necessary for structural interactions (particularly hydrogen bonds) with proteins, and typically include at least one amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two functional chemical groups. Candidate agents typically include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found in biomolecules, including peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Typically, multiple assay mixtures are run in parallel with different reagent concentrations to obtain different responses to different concentrations. Typically, one of these concentrations is used as a negative control, i.e., a concentration of zero or below the detection level.

[0109] "Full-length antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end. H), followed by a number of constant domains. Each light chain has a variable domain (V L ), and has 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 heavy chain variable domain. It is believed that specific amino acid residues form the interface between the light chain variable domain and the heavy chain variable domain.

[0110] An "isolated" molecule or cell is one that has been identified and separated from at least one contaminant 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 environment in which it is produced. The isolated molecule or cell is in a form that is different from its form or environment 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-C1s, anti-C1q, or anti-C1r antibody of the present disclosure. In other embodiments, the isolated cell is a host cell or hybridoma cell that produces an anti-C1s, anti-C1q, or anti-C1r antibody of the present disclosure.

[0111] An "isolated" antibody is one that has been identified, separated and / or recovered from a component of its production environment (e.g., naturally or recombinantly). Preferably, an isolated polypeptide is free from association with all other contaminating components from its production environment. Contaminating components from its production environment (e.g., from recombinant transfected cells) are substances that typically interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain preferred embodiments, the antibody is purified to the following extent: (1) greater than 95% by weight of the antibody, and most preferably greater than 99% by weight, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues by use of a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody includes the antibody in situ in a recombinant T cell, since at least one component of the antibody's natural environment will be absent. However, generally, an isolated polypeptide or antibody will be prepared by a method that includes at least one purification step.

[0112] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0113] The term "variable" refers to the fact that certain segments of the variable domain in an antibody are very different in sequence. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across the entire span of the variable domain. In contrast, it is concentrated in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, which mostly adopt a β-pleated structure and are connected by three HVRs, which form a loop connecting the β-sheet structure and sometimes form part of the β-sheet structure. The HVRs in each chain are tightly bound together by the FR region and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0114] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. By Kabat et al., J.Biol.Chem.252:6609-6616 (1977); Kabat et al., U.S. Department 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) CDRs are described, wherein the definitions include overlapping or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein.

[0115] As used herein, the terms "CDR-L1," "CDR-L2," and "CDR-L3" refer to the first, second, and third CDRs, respectively, in the light chain variable region. As used herein, the terms "CDR-H1," "CDR-H2," and "CDR-H3" refer to the first, second, and third CDRs, respectively, in the heavy chain variable region. As used herein, the terms "CDR-1," "CDR-2," and "CDR-3" refer to the first, second, and third CDRs, respectively, of either chain variable region.

[0116] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies in the population are identical, except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may exist in small quantities. Monoclonal antibodies have a high degree of specificity (for a single antigenic site). In contrast to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on the antigen. Except for its specificity, monoclonal antibodies are advantageous because they are typically synthesized by hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of an antibody obtained as a substantially homogeneous antibody population and should not be construed as needing to produce the antibody by any ad hoc method. For example, monoclonal antibodies for use in accordance with the present disclosure can be prepared 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, 2nd ed. 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., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et 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 partial or complete human immunoglobulin loci or genes encoding 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. Patent 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).

[0117] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably to refer to an antibody in substantially intact form, as opposed to an antibody fragment or antibody derivative. Specifically, whole antibodies include those having heavy and light chains that include an Fc region. The constant region may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0118] An "antibody fragment" or "functional fragment" of an antibody comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of an intact antibody, or the F region of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; and linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)). Other examples of antibody fragments include antibody derivatives, such as single-chain antibody molecules, monovalent antibodies, and multispecific antibodies formed from antibody fragments.

[0119] An "antibody derivative" is any construct comprising the antigen-binding region of an antibody. Examples of antibody derivatives include single-chain antibody molecules, monovalent antibodies, and multispecific antibodies formed from antibody fragments.

[0120] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. The Fab fragment consists of an intact L chain and the variable region domain (V H ) and the first constant domain of a heavy chain (C H 1) Composition. In terms of antigen binding, each Fab fragment is monovalent, that is, it has a single antigen binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which is roughly equivalent 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 the C H The carboxyl terminus of the constant domain has several additional residues, including one or more cysteines from the antibody hinge region. Fab' in which the cysteine ​​residue(s) of the constant domain have free thiol groups is referred to herein as Fab'-SH. F(ab')2 antibody fragments were initially produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0121] The Fc fragment comprises the carboxyl-terminal portions of two H chains held together by disulfides.The effector functions of an antibody are determined by the sequence of the Fc region, which is also recognized by Fc receptors (FcR) found on certain cell types.

[0122] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc regions of heavy chain immunoglobulins may be different, human IgG heavy chain Fc regions are generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (according to the EU numbering system, residue 447) in the Fc region can be removed, for example, during production or purification of antibodies or by recombinantly engineered nucleic acids encoding the heavy chain of an antibody. Therefore, the composition of a complete antibody can include antibody populations from which all K447 residues are removed, antibody populations from which no K447 residue is removed, and antibody populations with and without an antibody mixture containing the K447 residue. Native sequence Fc regions suitable for antibodies of the present disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0123] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include 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.

[0124] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by 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 a parent polypeptide, for example, from about one to about ten amino acid substitutions, preferably from about one to about five amino acid substitutions, in the native sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein will preferably have at least about 80% homology to the native sequence Fc region and / or to the Fc region of a parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0125] "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. Furthermore, a preferred FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors, the FcγRII receptors including FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily 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 deHaas et al., J. Lab. Clin. Med. 126:330-41 (1995). Additional FcRs (including those identified in the future) are encompassed by the term "FcR." FcRs may also increase the serum half-life of antibodies.

[0126] The binding and serum half-life of high-affinity human FcRn binding polypeptides can be determined in vivo, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered polypeptides with variant Fc regions. WO 2004 / 42072 (Presta) describes antibody variants with enhanced or diminished FcR binding. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0127] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight, non-covalent association. Six hypervariable loops (3 loops each from the H chain and L chain) are derived from the folding of these two domains, which provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the entire binding site.

[0128] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide also comprises a V H With V L A polypeptide linker between the domains that enables the sFv to form the structure required for antigen binding. For a review of sFv, see Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0129] The term "diabody" refers to a H With V L Small antibody fragments are prepared by constructing sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the domains, which allows interchain pairing of the V domains rather than intrachain pairing, thereby generating bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the V domains of the two antibodies are H and V L The domains are present on different polypeptide chains. Diabodies are described in detail in, for example, EP 404,097; WO 1993 / 011161; WO / 2009 / 121948; WO / 2014 / 191493; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48 (1993).

[0130] As used herein, a "chimeric antibody" refers to an antibody (immunoglobulin) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, so long as it exhibits the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies of interest herein include Antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced, for example, by immunizing a macaque with an antigen of interest. As used herein, "humanized antibodies" are a subset of "chimeric antibodies"

[0131] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody that contains minimal sequences derived from a non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the HVR of the receptor are replaced with residues from the HVR of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) with the desired specificity, affinity, and / or ability. In some cases, the FR residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may be included in residues not found in the receptor antibody or in the donor antibody. These modifications can be made to further improve the performance of the antibody, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein 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 those of a human immunoglobulin sequence, but the FR region may include one or more individual FR residue substitutions that can improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is generally no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional 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.

[0132] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or an antibody prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also useful for preparing human monoclonal antibodies are the methods 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 modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled, such as an immunized xenogeneic mouse (referring to the XENOMOUSE TM For human antibodies generated by human B cell hybridoma technology, see, eg, US Patent Nos. 6,075,181 and 6,150,584. For human antibodies generated by human B cell hybridoma technology, see also, eg, Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006).

[0133] The term "hypervariable region", "HVR" or "HV" as used herein refers to the region of the antibody variable domain whose sequence is highly variable and / or forms a structurally defined loop. Typically, an antibody comprises six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the greatest diversity among the six HVRs, and H3 in particular is considered to play a unique role in conferring good specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, 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, eg, Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0134] Many HVR descriptions are being used and encompassed in this article. HVR as Kabat complementary determining region (CDR) is based on the variability of sequence, and is the most commonly used (Kabat et al., supra). Chothia refers to the position of the structural loop (Chothia and Lesk J.Mol.Biol.196:901-917 (1987)). AbM HVR represents a compromise between Kabat CDR and Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software. " Contact " HVR is based on the analysis of available complex crystal structures. The residues from each of these HVRs are as follows.

[0135]

[0136] HVRs may include the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, 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.

[0137] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0138] The phrase "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the antibody compilation in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion of a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues 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 "standard" Kabat numbering sequence.

[0139] When referring to the 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 Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to the residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is generally used (e.g., Kabat et al., EU index reported in supra). The "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise indicated herein, reference to the residue numbering in the antibody variable domain refers to the residue numbering by the Kabat numbering system. Unless otherwise indicated herein, reference to the residue numbering in the antibody constant domain refers to the residue numbering by the EU numbering system (see, e.g., U.S. Patent Publication No. 2010-280227).

[0140] As used herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes 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. When pre-existing amino acid changes are present in VH, it is preferred that these changes occur at only three, two, or one of positions 71H, 73H, and 78H; for example, the amino acid residues at these positions may be 71A, 73T, and / or 78A. In some embodiments, the VL acceptor human framework sequence is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0141] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the subset of sequences is a subset as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include that for VL, the subgroup may be subgroup κI, κII, κIII, or κIV as in Kabat et al., supra. Additionally, for VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.

[0142] An "amino acid modification" at a specific position refers to a substitution or deletion of a specific residue, or the insertion of at least one amino acid residue near a specific residue. An insertion "adjacent" a specific residue refers to an insertion within one to two residues thereof. The insertion can be at the N-terminus or C-terminus of the specific residue. The preferred amino acid modification herein is a substitution.

[0143] An "affinity matured" antibody refers to an antibody with one or more changes in one or more of its HVRs that result in an increase in the antibody's affinity for the antigen compared to a parent antibody without those changes. In some embodiments, affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by methods known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and / or framework residues is described, for example, in: 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).

[0144] As used herein, the term "specific recognition" or "specific binding" refers to a measurable and repeatable interaction, such as attraction or binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically or preferentially binds to a target or epitope is one that binds to the target or epitope with greater affinity, avidity, more readily, and / or for a longer duration than it 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. Thus, "specific binding" or "preferential binding" does not necessarily require (but it can 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 cases about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 to 10 9 M -1 or about 10 10 M -1 to 10 11 M -1or higher association constant. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive with a protein. 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.

[0145] As used herein, "identity" means that at any particular position in the aligned sequences, the amino acid residues between the sequences are identical. As used herein, "similarity" indicates that at any particular position in the aligned sequences, the amino acid residues between the sequences are of similar type. For example, leucine can replace isoleucine or valine. Other amino acids that can often be substituted for each other include, but are not limited to:

[0146] - Phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains);

[0147] - lysine, arginine, and histidine (amino acids with basic side chains);

[0148] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0149] - Asparagine and glutamine (amino acids with amide side chains); and

[0150] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0151] Identity and similarity can be readily calculated (see, for example, 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).

[0152] As used herein, an "interaction" between a complement protein and a second protein encompasses, 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 it 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 it binds to one of the two proteins.

[0153] A "blocking" antibody, "antagonist" antibody, "inhibitory" antibody, or "neutralizing" antibody is an antibody that inhibits or reduces one or more biological activities of the antigen to which it binds, such as interaction with one or more proteins. 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 the antigen.

[0154] The term "inhibitor" refers to a compound that can inhibit the biological function of a target biomolecule (e.g., mRNA or protein) by reducing the activity or expression of the target biomolecule. Inhibitors can be antibodies, small molecules, or nucleic acid molecules. The term "antagonist" refers to a compound that binds to a receptor and blocks or inhibits the biological response of the receptor. The term "inhibitor" may also refer to "antagonist"

[0155] 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.

[0156] As used herein, the term "affinity" refers to the equilibrium constant that two reagents (for example, antibody and antigen) are reversibly combined, and is expressed as a dissociation constant (KD). Affinity can be at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times or at least 1,000 times or more than the affinity of the antibody to an unrelated amino acid sequence. The affinity of the antibody to the target protein can be, for example, about 100 nanomoles (nM) to about 0.1nM, about 100nM to about 1 picomoles (pM) or about 100nM to about 1 femtomoles (fM) or higher. As used herein, the term "affinity" refers to the resistance of a complex of two or more agents to dissociate upon dilution. With respect to antibodies and / or antigen-binding fragments, the terms "immunoreactivity" and "preferential binding" are used interchangeably herein.

[0157] 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. For example, a subject anti-C1s antibody specifically binds to an epitope within the complement C1s protein. "Specific binding" refers to an affinity of at least about 10 -7 M or larger, such as 5×10 -7 M, 10 -8 M, 5×10 -8 M and greater. "Non-specific binding" refers to an affinity less than about 10 -7 M, for example, with 10 -6 M, 10 -5 M, 10 -4 M etc. affinity binding.

[0158] The term "k on ” refers to the rate constant for the association of antibody with antigen.

[0159] The term "k off ” refers to the rate constant for the dissociation of an antibody from the antibody / antigen complex.

[0160] The term "K D ” refers to the equilibrium dissociation constant of the antibody-antigen interaction.

[0161] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a particular peptide or polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to achieve the maximum percent sequence identity, and not taking into account any conservative substitutions as part of the sequence identity. Alignment for 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.

[0162] "Biological sample" covers a variety of sample types obtained from an individual and can be used for diagnosis or monitoring determination. The definition covers liquid samples of blood and other biological origins, solid tissue samples (such as biopsy specimens or tissue cultures or cells derived therefrom) and their offspring. The definition also includes samples processed in any way after acquisition, such as by treating with reagents, dissolving or enriching certain components, such as polynucleotides. The term "biological sample" covers clinical samples, and also includes cultured cells, cell supernatants, cell lysates, serum, blood plasma, biological fluids and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, eye fluid, synovial fluid, blood components (such as blood plasma and serum) etc. The term "biological sample" also includes solid tissue samples, tissue culture samples and cell samples.

[0163] 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, an isolated nucleic acid is free from all components associated with the environment in which it is produced. An isolated nucleic acid molecule encoding the polypeptides and antibodies herein is in a form different from the form or environment in which it is found in nature. Thus, an isolated nucleic acid molecule is distinct from nucleic acids encoding any polypeptide or antibody naturally occurring in a cell.

[0164] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting other nucleic acids connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA to which an additional DNA segment can be connected. Another type of vector is a phage vector. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell they are introduced into (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to as "recombinant expression vectors" or simply "expression vectors" in this article. Generally, expression vectors useful in recombinant DNA technology are generally in the form of plasmids. In this manual, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms.

[0165] "Polynucleotide" or "nucleic acid" used interchangeably herein refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthesis reaction. The polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, the nucleotide structure can be modified before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide can contain modifications made after synthesis, such as in conjunction with a label. Other types of modifications include, for example, "caps" (substitution of one or more naturally occurring nucleotides with analogs); internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.); those containing side groups, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.); those with intercalating agents (e.g., acridine, psoralen, etc.); those containing chelating agents (e.g., metals, radioactive metals, boron, oxidative metals, etc.); those containing alkylating agents; those with modified bonds (e.g., alpha anomeric nucleic acids, etc.); and unmodified forms of polynucleotides. In addition, any hydroxyl group normally present in the sugar can be replaced with, for example, a phosphonate group, a phosphate group, protected with standard protecting groups, or activated to prepare additional linkages with additional nucleotides, or can be bound to a solid or semi-solid support. 5' and 3' terminal OH can be phosphorylated or partially replaced by amine or organic blocking groups of 1 to 20 carbon atoms. Other hydroxyls can also be derived into 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 (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 ("thioester"), P(S)S ("dithioester"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("methyl acetal"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl group optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical.The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0166] As used herein, "gene editing agents" are defined as agents for gene editing, representative examples of which include CRISPR-associated nucleases, such as Cas9 and Cpfl gRNAs, Argonaute endonuclease family, clustered regularly interspaced short palindromic repeats (CRISPR) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), large nucleases, other endonucleases and / or exonucleases. See Schiffer, 2012, J Virol 88(17):8920-8936, incorporated herein by reference.

[0167] As used herein, "RNA interference agent" is defined as any agent that interferes with or inhibits the expression of a target biomarker gene through RNA interference (RNAi). Such RNA interference agents include, but are not limited to, nucleic acid molecules, including RNA molecules homologous to the target biomarker gene of the present invention or fragments thereof, short interfering RNA (siRNA), and small molecules that interfere with or inhibit the expression of a target biomarker nucleic acid through RNA interference (RNAi).

[0168] "RNA interference (RNAi)" is an evolutionarily conserved process in which the expression or introduction of RNA with a sequence identical or highly similar to a target biomarker nucleic acid results in sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from the target gene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting the expression of the target biomarker nucleic acid. In one embodiment, the RNA is double-stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific nuclease Dicer, which promotes the processive cleavage of long dsRNAs into double-stranded fragments called siRNAs. The siRNAs are incorporated into protein complexes that recognize and cleave the target mRNA. RNAi can also be initiated by the introduction of nucleic acid molecules, such as synthetic siRNAs, shRNAs, or other RNA interfering agents, to inhibit or silence the expression of target biomarker nucleic acids. As used herein, "inhibition of target biomarker nucleic acid expression" or "inhibition of marker gene expression" includes any reduction in the expression of a target biomarker nucleic acid or a protein encoded by a target biomarker nucleic acid, or the activity or level of a protein. The reduction can be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to the expression of a target biomarker nucleic acid that is not targeted by an RNA interfering agent, or the activity or level of a protein encoded by a target biomarker nucleic acid that is not targeted by an RNA interfering agent.

[0169] In addition to RNAi, genome editing can be used to regulate the copy number or genetic sequence of a biomarker of interest, such as constitutive or inducible knockout or mutation of a biomarker of interest, such as complement pathway components such as C1q, C1r and / or C1s. For example, the CRISPR-Cas system can be used to precisely edit genomic nucleic acids (e.g., for creating non-functional or invalid mutations). In such embodiments, CRISPR guide RNA and / or Cas enzymes can be expressed. For example, a vector containing only guide RNA can be administered to an animal or cell transgenic for the Cas9 enzyme. Similar strategies can be used (e.g., designing zinc fingers, transcription activator-like effectors (TALEs), or homing large nucleases). Such systems are well known in the art (see, e.g., U.S. Patent No. 8,697,359; Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Patent Publications 2014 / 0087426 and 2012 / 0178169; Boch et al. (2011) Nat. Biotech. 29:135-136; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al. (2011) PLoS One6:e19722; Li et al. (2011) Nucl. Acids Res. 39:6315-6325; Zhang et al. (2011) Nat. Biotech. 29:149-153; Miller et al. (2011) Nat. Biotech. 29:143-148; Lin et al. (2014) Nucl. Acids Res. 42:e47). Such genetic strategies can use constitutive expression systems or inducible expression systems according to methods well known in the art.

[0170] "Piwi-interacting RNA (piRNA)" is the largest class of non-coding small RNA molecules. piRNAs form RNA-protein complexes by interacting with piwi proteins. These piRNA complexes are associated with epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germline cells (particularly germline cells during spermatogenesis). They differ from microRNAs (miRNAs) in size (26-31nt instead of 21-24nt), lack of sequence conservation, and increased complexity. However, like other small RNAs, piRNAs are thought to be involved in gene silencing, particularly the silencing of transposons. Most piRNAs are antisense to transposon sequences, indicating that transposons are piRNA targets. In mammals, piRNA activity in transposon silencing appears to be most important during embryonic development, and in both Caenorhabditis elegans and humans, piRNAs are essential for spermatogenesis. piRNAs play a role in RNA silencing by forming an RNA-induced silencing complex (RISC).

[0171] "Aptamers" are oligonucleotides or peptide molecules that bind to specific target molecules. "Nucleic acid aptamers" are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalent SELEX (systematic evolution of ligand exponential enrichment) to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. "Peptide aptamers" are artificial proteins that are selected or engineered to bind to specific target molecules. These proteins consist of one or more variable sequence peptide loops displayed by a protein scaffold. They are usually isolated from combinatorial libraries and are usually subsequently improved by directed mutagenesis or multiple rounds of variable region mutagenesis and selection. "Affibin" is the evolution of peptide aptamers, which are small and highly stable proteins designed to display peptide loops and provide a high-affinity binding surface for a specific target protein. It is a low molecular weight protein with a molecular weight of 12-14 kDa and is derived from the cysteine ​​protease inhibitor family. Aptamers are useful in biotechnology and therapeutic applications because they provide molecular recognition properties comparable to commonly used biomolecule antibodies. In addition to their discriminatory recognition, aptamers offer advantages over antibodies in that they can be engineered entirely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.

[0172] "Short interfering RNA" (siRNA), also referred to herein as "small interfering RNA", is defined as an agent that inhibits the expression of a target biomarker nucleic acid, for example, by RNAi. siRNA can be chemically synthesized, can be produced by in vitro transcription, or can be produced within a host cell. In one embodiment, the siRNA is a double-stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides in length, more preferably about 19 to about 25 nucleotides in length, more preferably about 19, 20, 21 or 22 nucleotides in length, and can contain 3' and / or 5' overhangs of about 0, 1, 2, 3, 4 or 5 nucleotides in length on each strand. The length of the overhangs is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Preferably, the siRNA is capable of promoting RNA interference by degradation of the target messenger RNA (mRNA) or specific post-transcriptional gene silencing (PTGS).

[0173] In another embodiment, siRNA is a small hairpin (also called stem-loop) RNA (shRNA). In one embodiment, these shRNAs consist of a short (e.g., 19-25 nucleotide) antisense strand, followed by a loop of 5-9 nucleotides, and a similar sense strand. Alternatively, the sense strand can be preceded by the nucleotide loop structure and the antisense strand can be followed. These shRNAs can be contained in plasmids, retroviruses, and lentiviruses and expressed by, for example, pol III U6 promoter or another promoter (see, e.g., Stewart et al. (2003) RNA Apr; 9(4): 493-501, incorporated herein by reference).

[0174] "Host cell" includes a single cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny are not necessarily completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with a polynucleotide of the present disclosure.

[0175] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals exposed thereto at the dosages and concentrations used. Typically, a physiologically acceptable carrier is a pH-buffered aqueous 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 ).

[0176] The term "prevention" is art-recognized and is well known in the art when used for conditions such as epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy), and includes the administration of a composition that reduces the frequency or severity of, or delays the onset of, one or more symptoms of the medical condition in a subject relative to a subject who does not receive the composition. Similarly, prevention of epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy) includes reducing the likelihood that a patient receiving treatment (relative to a patient not receiving treatment) will develop epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy).

[0177] As used herein, the term "subject" refers to a living mammal and is 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 monkey species); agricultural animals (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 denote a particular age or sex.

[0178] As used herein, the terms "treating" or "treatment" include alleviating a condition, arresting a condition, or reversing the symptoms, clinical signs, or underlying pathology of a condition, to stabilize or improve a subject's condition, or to reduce the likelihood that a subject's condition will worsen, as if the subject were not receiving treatment.

[0179] The term "therapeutically effective amount" of a compound with respect to the subject methods of treatment refers to that amount of the compound in a formulation that, when administered as part of a desired dosing regimen (to a mammal, preferably a human), alleviates symptoms, ameliorates symptoms, or slows the onset of a disease condition, e.g., at a reasonable benefit / risk ratio applicable to any drug therapy, according to clinically acceptable standards for the disorder or condition being treated or for cosmetic purposes. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, as well as the ability of the antibody to elicit a desired response in the individual.

[0180] As used herein, an individual who is "at risk" of developing a particular disease, disorder, or condition may or may not have detectable disease or disease symptoms, and may or may not have detectable disease or disease symptoms prior to the methods of treatment described herein. "At risk" means that an individual has one or more risk factors, which are measurable parameters (as known in the art) associated with the development of a particular disease, disorder, or condition. An individual with one or more of these risk factors is more likely to develop a particular disease, disorder, or condition than an individual without one or more of these risk factors.

[0181] "Chronic" administration refers to administration in a continuous rather than acute manner, thereby maintaining the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration refers to treatment that is not continuously administered without interruption, but is cyclic / periodic in nature.

[0182] As used herein, administration "in conjunction" with another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a common co-formulation or administration as separate compositions, including different dosing frequencies or intervals, and using the same route of administration or different routes of administration.

[0183] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with the cited publications. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (ed. FMAusubel et al. (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (edited by MJMacPherson, BD Hames and GRTaylor (1995)), Harlow and Lane (edited (1988)) Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by RI Freshney (1987)); Oligonucleotide Synthesis (edited by MJGait, 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (edited by JECellis, 1998) Academic Press; Animal CellCulture (RI Freshney), 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, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CCBlackwell (ed.); GeneTransfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wiley and et al., 1991); Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty, IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory The widely used methods are described in The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0184] Anti-complement C1q antibodies

[0185] The anti-C1q antibodies disclosed herein are potent inhibitors of C1q and can be administered for any period of time to provide sustained inhibition of the peripheral and central nervous systems, then optionally discontinued to allow restoration of normal C1q function when its activity may be important for central nervous system repair. Results obtained in animal studies with the anti-C1q antibodies disclosed herein can be readily brought to the clinic with humanized versions of the same antibodies (the antibodies disclosed herein cross-react with mouse and human C1q), as well as fragments and / or derivatives thereof.

[0186] C1q is a 460 kDa polymeric protein composed 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 to form the C1 complex (C1qr2s2).

[0187] Suitable inhibitors include antibodies that bind complement factor C1q and / or C1q in the C1 complex of the classical complement activation pathway. The binding complement factor can be derived from, but is not limited to, any organism with a complement system, including any mammalian organism, such as humans, mice, rats, rabbits, monkeys, dogs, cats, cows, horses, camels, sheep, goats, or pigs.

[0188] As used herein, "C1 complex" refers to a protein complex that may include, but is not limited to, one C1q protein, two C1r proteins, and two C1s proteins (e.g., C1qr 2 s 2 ).

[0189] As used herein, "complement factor C1q" refers to the wild-type sequence and naturally occurring variant sequences.

[0190] A non-limiting example of a complement factor C1q recognized by the antibodies of the present disclosure is human C1q, comprising three polypeptide chains A, B, and C:

[0191] C1q, chain A (Homo sapiens), accession number protein

[0192] Database: NP_057075.1; GenBank ID: NM_015991:

[0193] >gi|7705753|ref|NP_057075.1|Component C1q

[0194] Subcomponent subunit A precursor [Homo sapiens]

[0195] (SEQ ID NO: 1)

[0196] MEGPRGWLVLCVLAISLASMVTEDLCCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRN PPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA.

[0197] C1q, chain B (Homo sapiens), accession number protein

[0198] Database: NP_000482.3; GenBank ID: NM_000491.3:

[0199] >gi|87298828|ref|NP_000482.3|Component Clq

[0200] Subcomponent subunit B precursor [Homo sapiens]

[0201] (SEQ ID NO: 2)

[0202] MMMKIPWGSIPVLMLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSAT RTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA.

[0203] C1q, chain C (Homo sapiens), accession number protein

[0204] Database: NP_001107573.1; GenBank ID:

[0205] NM_001114101.1:

[0206] >gi|166235903|ref|NP_001107573.1|Component C1q

[0207] Subunit C precursor [Homo sapiens]

[0208] (SEQ ID NO:3)

[0209] MDVGPPSSLPHLGLKLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDGLPPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQSVFT VTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD.

[0210] Thus, the anti-C1q antibodies of the present disclosure can bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of the C1q protein. In some embodiments, the anti-C1q antibodies of the present disclosure bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of human C1q or a homolog thereof, such as mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig C1q. In some embodiments, the anti-C1q antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0211] Suitable antibodies include antibodies that bind to complement C1q protein (i.e., anti-complement C1q antibodies, also referred to herein as anti-C1q antibodies and C1q antibodies) and nucleic acid molecules encoding such antibodies for use in methods for preventing, reducing the risk of developing epilepsy, or treating epilepsy, such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy.

[0212] Other anti-C1q antibodies suitable for binding to C1q protein are well known in the art and include, for example, antibodies Cat#: AF2379, AF1696, MAB1696, and MAB23791 (R&D System); NBP1-87492, NB100-64420, H00000712-B01P, H00000712-D01P, and H00000712-D01 (Novus Biologicals); MA1-83963, MA1-40311, PA5-14208, PA5-29586, and PA1-36177 (ThermoFisher Scientific); ab71940, ab11861, ab4223, ab72355, ab182451, ab46191, ab227072, ab182940, ab216979, and ab235454 (abcam), etc. In addition, a variety of siRNA, shRNA, and CRISPR constructs for reducing C1q expression can be found in the commercial product lists of the above-mentioned companies, such as SiRNA products #sc-43651, sc-44962, sc-105153, and sc-141842; ShRNA products #sc-43651-SH, sc-43651-V, sc-44962-SH, sc-44962-V, sc-105153-SH, sc-105153-V, sc-141842-SH, and sc-14184 2-V; CRISPR Products #sc-419385, sc-419385-HDR, sc-419385-NIC, sc-419385-NIC-2, sc-402156, sc-402156-KO-2, sc-404309, sc-404309-HDR, sc-404309-NIC, sc-404309-NIC-2, sc-419386, sc-419386-HDR, sc-419386-NIC, sc-419386-NIC-2 (Santa Cruz Biotechnology et al.).

[0213] All sequences mentioned in the following twenty paragraphs are incorporated by reference into US Patent No. 9,708,394, the disclosure of which is incorporated by reference into antibodies and related compositions.

[0214] Light chain and heavy chain variable domain sequences of antibody M1

[0215] Using standard techniques, the nucleic acid and amino acid sequences encoding the light chain variable domain and heavy chain variable domain of antibody M1 were determined. The amino acid sequence of the light chain variable domain of antibody M1 is:

[0216]

[0217] The hypervariable regions (HVRs) of the light chain variable domains are in bold and underlined. In some embodiments, HVR-L1 of the M1 light chain variable domain has the sequence RASKSINKYLA (SEQ ID NO: 5), HVR-L2 of the M1 light chain variable domain has the sequence SGSTLQS (SEQ ID NO: 6), and HVR-L3 of the M1 light chain variable domain has the sequence QQHNEYPLT (SEQ ID NO: 7).

[0218] The amino acid sequence of the heavy chain variable domain of antibody M1 is:

[0219]

[0220] The hypervariable region (HVR) of the heavy chain variable domain is bold and underlined. In some embodiments, the HVR-H1 of the M1 heavy chain variable domain has the sequence GYHFTSYWMH (SEQ ID NO: 9), the HVR-H2 of the M1 heavy chain variable domain has the sequence VIHPNSGSINYNEKFES (SEQ ID NO: 10), and the HVR-H3 of the M1 heavy chain variable domain has the sequence ERDSTEVLPMDY (SEQ ID NO: 11).

[0221] The nucleic acid sequence encoding the light chain variable domain was determined to be:

[0222] GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAACAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTG CAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAACATAATGAATACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:12).

[0223] The nucleic acid sequence encoding the heavy chain variable domain was determined to be:

[0224] CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACCATTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGTGATTCATCCTAATAGTGGTAGTATTAACTACAAT GAGAAGTTCGAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCGGCGGTCTATTATTGTGCAGGAGAGAGAGATTCTACGGAGGTTCTCCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ IDNO:13).

[0225] Material Preservation

[0226] The following materials have been deposited under the Budapest Treaty with the American Type Culture Collection, ATCC Patent Depository, 10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC):

[0227]

[0228] The hybridoma cell line producing the M1 antibody (mouse hybridoma C1qM1 7788-1(M)051613) has been deposited with the ATCC under conditions ensuring that cultures will be available during the pendency of the patent application for a period of 30 years, or 5 years after the most recent request, or the effective life of the patent, whichever is longer. If the deposit becomes unavailable during this period, the deposit will be replaced. The deposit is available as required by foreign patent laws in countries in which counterparts or progeny of the subject application are filed. However, it should be understood that the availability of the deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

[0229] Disclosed herein are methods of administering an anti-C1q antibody comprising a light chain variable domain and a heavy chain variable domain. The antibody may bind to at least human C1q, mouse C1q, or rat C1q. The antibody may be a humanized antibody, a chimeric antibody, or a human antibody. The antibody may be a monoclonal antibody, an antibody fragment thereof, and / or an antibody derivative thereof. The light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3 of the monoclonal antibody M1 produced by the hybridoma cell line deposited with Accession No. PTA-120399. The heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody M1 produced by the hybridoma cell line deposited with ATCC Accession No. PTA-120399.

[0230] In some embodiments, the amino acid sequences of the light chain variable domain and the heavy chain variable domain comprise one or more of SEQ ID NO: 5 of HVR-L1, SEQ ID NO: 6 of HVR-L2, SEQ ID NO: 7 of HVR-L3, SEQ ID NO: 9 of HVR-H1, SEQ ID NO: 10 of HVR-H2, and SEQ ID NO: 11 of HVR-H3.

[0231] The antibody may comprise a light chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 4, preferably while retaining HVR-L1 RASKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6) and HVR-L3 QQHNEYPLT (SEQ ID NO: 7). The antibody may comprise a heavy chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 8, preferably while retaining HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10) and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0232] Disclosed herein are methods of administering anti-Clq antibodies that inhibit the interaction between Clq and autoantibodies. In preferred embodiments, the anti-Clq antibodies promote clearance of Clq from circulation or tissues.

[0233] The anti-C1q antibody can bind to the C1q protein and bind to one or more amino acids of the C1q protein at an amino acid residue selected from the group consisting of: (a) amino acid residues 196-226 of SEQ ID NO: 1 (SEQ ID NO: 16), or amino acid residues of C1q protein chain A (C1qA) corresponding to amino acid residues 196-226 of SEQ ID NO: 1 (GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) (SEQ ID NO: 16); (b) amino acid residues 196-221 of SEQ ID NO: 1 (SEQ ID NO: 17), or amino acid residues of C1qA corresponding to amino acid residues 196-221 of SEQ ID NO: 1 (GLFQVVSGGMVLQLQQGDQVWVEKDP) (SEQ ID NO: 17); (c) amino acid residues 202-221 of SEQ ID NO: 1 (SEQ ID NO: 18), or amino acid residues corresponding to amino acid residues 203-221 of SEQ ID NO: 1 (SEQ ID NO: 19). NO:1 (SGGMVLQLQQGDQVWVEKDP) (SEQ ID NO: 18); (d) amino acid residues 202-219 of SEQ ID NO: 1 (SEQ ID NO: 19), or amino acid residues of C1qA corresponding to amino acid residues 202-219 of SEQ ID NO: 1 (SGGMVLQLQQGDQVWVEK) (SEQ ID NO: 19); and (e) amino acid residues Lys 219 and / or Ser 202 of SEQ ID NO: 1, or amino acid residues of C1qA corresponding to Lys 219 and / or Ser 202 of SEQ ID NO: 1.

[0234] In some embodiments, the antibody further binds to one or more amino acids of the C1q protein at an amino acid residue selected from the group consisting of: (a) amino acid residues 218-240 of SEQ ID NO: 3 (SEQ ID NO: 20), or amino acid residues of C1q protein chain C (C1qC) corresponding to amino acid residues 218-240 of SEQ ID NO: 3 (WLAVNDYYDMVGI QGSDSVFSGF) (SEQ ID NO: 20); (b) amino acid residues 225-240 of SEQ ID NO: 3 (SEQ ID NO: 21), or amino acid residues of C1qC corresponding to amino acid residues 225-240 of SEQ ID NO: 3 (YDMVGI QGSDSVFSGF) (SEQ ID NO: 21); (c) amino acid residues 225-232 of SEQ ID NO: 3 (SEQ ID NO: 22), or amino acid residues of C1q corresponding to amino acid residues 225-232 of SEQ ID NO: 3 (YDMVGIQG) (SEQ ID NO: (d) amino acid residue Tyr 225 of SEQ ID NO: 3, or an amino acid residue of C1qC corresponding to amino acid residue Tyr 225 of SEQ ID NO: 3; (e) amino acid residues 174-196 of SEQ ID NO: 3 (SEQ ID NO: 23), or an amino acid residue of C1qC corresponding to amino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO: 3 (SEQ ID NO: 23); (f) amino acid residues 184-192 of SEQ ID NO: 3 (SEQ ID NO: 24), or an amino acid residue of C1qC corresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO: 3 (SEQ ID NO: 24); (g) amino acid residues 185-187 of SEQ ID NO: 3, or an amino acid residue of C1qC corresponding to amino acid residues 185-187 of SEQ ID NO: 3 (SEQ ID NO: 23); amino acid residues 185-187 (SGV) of C1qC of SEQ ID NO:3; (h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acid residue of C1qC corresponding to amino acid residue Ser 185 of SEQ ID NO:3.

[0235] In certain embodiments, an anti-C1q antibody binds to amino acid residues Lys219 and Ser 202 of human C1qA as set forth in SEQ ID NO: 1, or amino acids corresponding to Lys 219 and Ser 202 of human C1qA as set forth in SEQ ID NO: 1; and amino acid residue Tyr 225 of human C1qC as set forth in SEQ ID NO: 3, or an amino acid residue of human C1qC corresponding to Tyr 225 as set forth in SEQ ID NO: 3. In certain embodiments, an anti-C1q antibody binds to amino acid residue Lys 219 of human C1qA as set forth in SEQ ID NO: 1, or an amino acid residue of human C1qA corresponding to Lys 219 as set forth in SEQ ID NO: 1; and amino acid residue Ser 185 of human C1qC as set forth in SEQ ID NO: 3, or an amino acid residue of human C1qC corresponding to Ser 185 as set forth in SEQ ID NO: 3.

[0236] In some embodiments, the anti-C1q antibody binds to the C1q protein and binds to one or more amino acids of the C1q protein at an amino acid residue selected from the group consisting of: (a) amino acid residues 218-240 of SEQ ID NO:3 (SEQ ID NO:20), or amino acid residues of C1qC corresponding to amino acid residues 218-240 of SEQ ID NO:3 (WLAVNDYYDMVGI QGSDSVFSGF) (SEQ ID NO:20); (b) amino acid residues 225-240 of SEQ ID NO:3 (SEQ ID NO:21), or amino acid residues of C1qC corresponding to amino acid residues 225-240 of SEQ ID NO:3 (YDMVGI QGSDSVFSGF) (SEQ ID NO:21); (c) amino acid residues 225-232 of SEQ ID NO:3 (SEQ ID NO:22), or amino acid residues of C1qC corresponding to amino acid residues 225-232 of SEQ ID NO:3 (YDMVGIQG) (SEQ ID NO: (d) amino acid residue Tyr 225 of SEQ ID NO: 3, or an amino acid residue of C1qC corresponding to amino acid residue Tyr 225 of SEQ ID NO: 3; (e) amino acid residues 174-196 of SEQ ID NO: 3 (SEQ ID NO: 23), or an amino acid residue of C1qC corresponding to amino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO: 3 (SEQ ID NO: 23); (f) amino acid residues 184-192 of SEQ ID NO: 3 (SEQ ID NO: 24), or an amino acid residue of C1qC corresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO: 3 (SEQ ID NO: 24); (g) amino acid residues 185-187 of SEQ ID NO: 3, or an amino acid residue of C1qC corresponding to amino acid residues 185-187 of SEQ ID NO: 3 (SEQ ID NO: 23); amino acid residues 185-187 (SGV) of C1qC of SEQ ID NO:3; (h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acid residue of C1qC corresponding to amino acid residue Ser 185 of SEQ ID NO:3.

[0237] In some embodiments, the anti-C1q antibodies of the present disclosure inhibit the interaction between C1q and C1s. In some embodiments, the anti-C1q antibodies inhibit the interaction between C1q and C1r. In some embodiments, the anti-C1q antibodies inhibit the interaction between C1q and C1s and between C1q and C1r. In some embodiments, the anti-C1q antibodies inhibit the interaction between C1q and another antibody (e.g., an autoantibody). In preferred embodiments, the anti-C1q antibodies promote the clearance of C1q from the circulation or tissues. In some embodiments, the anti-C1q antibodies inhibit the respective interactions at a stoichiometry of less than 2.5:1, 2.0:1, 1.5:1, or 1.0:1. In some embodiments, at approximately equimolar concentrations of C1q and anti-C1q antibody, the C1q antibody inhibits an interaction, such as a C1q-C1s interaction. In other embodiments, the anti-C1q antibody is expressed in a ratio of less than 20:1; less than 19.5:1; less than 19:1; less than 18.5:1; less than 18:1; less than 17.5:1; less than 17:1; less than 16.5:1; less than 16:1; less than 15.5:1; less than 15:1; less than 14.5:1; less than 14:1; less than 13.5:1; less than 13:1; less than 12.5:1; less than 12:1; less than 11.5:1; less than In some embodiments, the anti-C1q antibody binds to C1q at a stoichiometry of less than 11:1; less than 10.5:1; less than 10:1; less than 9.5:1; less than 9:1; less than 8.5:1; less than 8:1; less than 7.5:1; less than 7:1; less than 6.5:1; less than 6:1; less than 5.5:1; less than 5:1; less than 4.5:1; less than 4:1; less than 3.5:1; less than 3:1; less than 2.5:1; less than 2.0:1; less than 1.5:1; or less than 1.0: 1. ....0:1. In certain embodiments, an anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 2.5:1 to 1.0:1 or less than 1.0:1. In some embodiments, an anti-C1q antibody inhibits the interaction between C1q and C1r, between C1q and C1s, or between C1q and both C1r and C1s. In some embodiments, an anti-C1q antibody inhibits the interaction between C1q and C1r, between C1q and C1s, and / or between C1q and both C1r and C1s. In some embodiments, an anti-C1q antibody binds to C1q A chain. In other embodiments, an anti-C1q antibody binds to C1q B chain. In other embodiments, an anti-C1q antibody binds to C1q C chain.In some embodiments, the anti-C1q antibody binds to the C1q A chain, C1q B chain, and / or C1q C chain. In some embodiments, the anti-C1q antibody binds to the globular domain of the C1q A chain, C1q B chain, and / or C chain. In other embodiments, the anti-C1q antibody binds to the collagen-like domain of the C1q A chain, C1q B chain, and / or C1q C chain.

[0238] When an antibody of the present disclosure inhibits an interaction between two or more complement factors, such as the interaction between C1q and C1s or the interaction between C1q and C1r, the interaction that occurs in the presence of the antibody can be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% relative to a control in which the antibody of the present disclosure is not present. In certain embodiments, the interaction that occurs in the presence of the antibody is reduced by an amount ranging from at least 30% to at least 99% relative to a control in which the antibody of the present disclosure is not present.

[0239] In some embodiments, the antibodies of the present disclosure inhibit C2 or C4 cleavage by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or by an amount ranging from at least 30% to at least 99%, relative to a control in which the antibodies of the present disclosure are not present. Methods for measuring C2 or C4 cleavage are well known in the art. The EC values ​​of the antibodies of the present disclosure for C2 or C4 cleavage are as follows: 50 Values ​​may be less than 3 μg / ml; 2.5 μg / ml; 2.0 μg / ml; 1.5 μg / ml; 1.0 μg / ml; 0.5 μg / ml; 0.25 μg / ml; 0.1 μg / ml; 0.05 μg / ml. In some embodiments, the antibodies of the present disclosure inhibit C2 or C4 cleavage at approximately equimolar concentrations of CIq and the corresponding anti-CIq antibody.

[0240] In some embodiments, the antibodies of the present disclosure inhibit autoantibody-dependent and complement-dependent cytotoxicity (CDC) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or by an amount ranging from at least 30% to at least 99%, relative to a control in which the antibodies of the present disclosure are not present. 50Values ​​can be less than 3 μg / ml; 2.5 μg / ml; 2.0 μg / ml; 1.5 μg / ml; 1.0 μg / ml; 0.5 μg / ml; 0.25 μg / ml; 0.1 μg / ml; 0.05 μg / ml.

[0241] In some embodiments, the antibodies of the present disclosure inhibit complement dependent cell-mediated cytotoxicity (CDCC) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or by an amount ranging from at least 30% to at least 99%, relative to a control in which the antibodies of the present disclosure are not present. Methods for measuring CDCC are well known in the art. EC values ​​for CDCC inhibition by the antibodies of the present disclosure are as follows: 50 Values ​​may be less than 3 μg / ml; 2.5 μg / ml; 2.0 μg / ml; 1.5 μg / ml; 1.0 μg / ml; 0.5 μg / ml; 0.25 μg / ml; 0.1 μg / ml; 0.05 μg / ml. In some embodiments, the antibodies of the present disclosure inhibit CDCC but not antibody-dependent cellular cytotoxicity (ADCC).

[0242] Humanized anti-complement C1q antibody

[0243] The humanized antibodies disclosed herein specifically bind to complement factor C1q and / or C1q protein in the C1 complex of the classical complement pathway. Humanized anti-C1q antibodies can specifically bind to human C1q, human and mouse C1q, rat C1q, or human C1q, mouse C1q, and rat C1q.

[0244] All sequences mentioned in the following sixteen paragraphs are incorporated by reference into U.S. Patent No. 10,316,081, the disclosure of which is incorporated by reference into the disclosure of antibodies and related compositions.

[0245] In some embodiments, the human heavy chain constant region is a human IgG4 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 47, or having at least 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% homology to SEQ ID NO: 47. According to Kabat numbering, the human IgG4 heavy chain constant region may comprise an Fc region having one or more modifications and / or amino acid substitutions. In this case, the Fc region comprises an amino acid substitution of leucine to glutamic acid at position 248, wherein such substitution inhibits interaction of the Fc region with an Fc receptor. In some embodiments, the Fc region comprises an amino acid substitution of serine to proline at position 241, wherein such substitution prevents arm switching in the antibody.

[0246] The amino acid sequence of the human IgG4 (S241P L248E) heavy chain constant domain is:

[0247] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:47).

[0248] The antibody may comprise a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 31-34, or an amino acid sequence having at least about 90% homology to an amino acid sequence selected from any one of SEQ ID NOs: 31-34. In certain such embodiments, the light chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 35-38, or an amino acid sequence having at least about 90% homology to an amino acid sequence selected from any one of SEQ ID NOs: 35-38.

[0249] The amino acid sequence of heavy chain variable domain variant 1 (VH1) is:

[0250] The hypervariable regions (HVRs) of VH1 are in bold and underlined.

[0251] The amino acid sequence of heavy chain variable domain variant 2 (VH2) is:

[0252] The hypervariable region (HVR) of VH2 is in bold and underlined.

[0253] The amino acid sequence of heavy chain variable domain variant 3 (VH3) is:

[0254] The hypervariable region (HVR) of VH3 is in bold and underlined.

[0255] The amino acid sequence of heavy chain variable domain variant 4 (VH4) is:

[0256] The hypervariable region (HVR) of VH4 is bold and underlined.

[0257] The amino acid sequence of kappa light chain variable domain variant 1 (Vκ1) is:

[0258] The hypervariable region (HVR) of Vκ1 is bold and underlined.

[0259] The amino acid sequence of kappa light chain variable domain variant 2 (Vκ2) is:

[0260] The hypervariable region (HVR) of Vκ2 is bold and underlined.

[0261] The amino acid sequence of kappa light chain variable domain variant 3 (Vκ3) is:

[0262] The hypervariable region (HVR) of Vκ3 is bold and underlined.

[0263] The amino acid sequence of kappa light chain variable domain variant 4 (Vκ4) is:

[0264] The hypervariable region (HVR) of Vκ4 is bold and underlined.

[0265] The antibody may comprise a light chain variable domain amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NOs: 35-38, while retaining HVR-L1 RASKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6), and HVR-L3 QQHNEYPLT (SEQ ID NO: 7). The antibody may comprise a heavy chain variable domain amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NOs: 31-34, while retaining HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0266] In some embodiments, the humanized anti-C1q antibodies of the present disclosure include a heavy chain variable region comprising a Fab region and a heavy chain constant region comprising an Fc region, wherein the Fab region specifically binds to the C1q protein of the present disclosure, but the Fc region cannot bind to the C1q protein. In some embodiments, the Fc region is from a human IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fc region cannot induce complement activity and / or cannot induce antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc region comprises one or more modifications, including but not limited to amino acid substitutions. In certain embodiments, the Fc region of the humanized anti-C1q antibodies of the present disclosure comprises an amino acid substitution at position 248 according to the Kabat numbering convention or a position corresponding to position 248 according to the Kabat numbering convention and / or at position 241 according to the Kabat numbering convention or a position corresponding to position 241 according to the Kabat numbering convention. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 inhibits the interaction of the Fc region with an Fc receptor. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 is an amino acid substitution from leucine to glutamic acid. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 prevents arm switching in the antibody. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 is an amino acid substitution from serine to proline. In certain embodiments, the Fc region of a humanized anti-C1q antibody of the present disclosure comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homology to the amino acid sequence of SEQ ID NO: 47.

[0267] In some embodiments, the humanized anti-C1q antibodies of the present disclosure can bind to the C1q protein and bind to one or more amino acids of the C1q protein at an amino acid residue selected from the group consisting of: (a) amino acid residues 196-226 of SEQ ID NO: 1 (SEQ ID NO: 16), or amino acid residues of C1q protein chain A (C1qA) corresponding to amino acid residues 196-226 of SEQ ID NO: 1 (GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) (SEQ ID NO: 16); (b) amino acid residues 196-221 of SEQ ID NO: 1 (SEQ ID NO: 17), or amino acid residues of C1qA corresponding to amino acid residues 196-221 of SEQ ID NO: 1 (GLFQVVSGGMVLQLQQGDQVWVEKDP) (SEQ ID NO: 17); (c) amino acid residues 202-221 of SEQ ID NO: 1 (SEQ ID NO: 18), or amino acid residues corresponding to SEQ ID NO: (d) amino acid residues 202-219 of SEQ ID NO: 1 (SEQ ID NO: 19), or amino acid residues of C1qA corresponding to amino acid residues 202-219 (SGGMVLQLQQGDQVWVEK) (SEQ ID NO: 19) of SEQ ID NO: 1; and (e) amino acid residues Lys 219 and / or Ser 202 of SEQ ID NO: 1, or amino acid residues of C1qA corresponding to Lys 219 and / or Ser 202 of SEQ ID NO: 1.

[0268] In some embodiments, the humanized anti-C1q antibody can further bind to one or more amino acids of the C1q protein at an amino acid residue selected from the group consisting of: (a) amino acid residues 218-240 of SEQ ID NO: 3 (SEQ ID NO: 20), or amino acid residues of C1q protein chain C (C1qC) corresponding to amino acid residues 218-240 of SEQ ID NO: 3 (WLAVNDYYDMVGI QGSDSVFSGF) (SEQ ID NO: 20); (b) amino acid residues 225-240 of SEQ ID NO: 3 (SEQ ID NO: 21), or amino acid residues of C1qC corresponding to amino acid residues 225-240 of SEQ ID NO: 3 (YDMVGI QGSDSVFSGF) (SEQ ID NO: 21); (c) amino acid residues 225-232 of SEQ ID NO: 3 (SEQ ID NO: 22), or amino acid residues of C1q corresponding to amino acid residues 225-232 of SEQ ID NO: 3 (YDMVGIQG) (SEQ ID NO: (d) amino acid residue Tyr 225 of SEQ ID NO: 3, or an amino acid residue of C1qC corresponding to amino acid residue Tyr 225 of SEQ ID NO: 3; (e) amino acid residues 174-196 of SEQ ID NO: 3 (SEQ ID NO: 23), or an amino acid residue of C1qC corresponding to amino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO: 3 (SEQ ID NO: 23); (f) amino acid residues 184-192 of SEQ ID NO: 3 (SEQ ID NO: 24), or an amino acid residue of C1qC corresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO: 3 (SEQ ID NO: 24); (g) amino acid residues 185-187 of SEQ ID NO: 3, or an amino acid residue of C1qC corresponding to amino acid residues 185-187 of SEQ ID NO: 3 (SEQ ID NO: 23); amino acid residues 185-187 (SGV) of C1qC of SEQ ID NO:3; (h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acid residue of C1qC corresponding to amino acid residue Ser 185 of SEQ ID NO:3.

[0269] In certain embodiments, the humanized anti-C1q antibodies of the present disclosure can bind to amino acid residues Lys 219 and Ser 202 of human C1qA as set forth in SEQ ID NO: 1, or amino acids corresponding to Lys 219 and Ser 202 of human C1qA as set forth in SEQ ID NO: 1; and amino acid residue Tyr 225 of human C1qC as set forth in SEQ ID NO: 3, or amino acid residues corresponding to Tyr 225 of human C1qC as set forth in SEQ ID NO: 3. In certain embodiments, the anti-C1q antibodies bind to amino acid residue Lys 219 of human C1qA as set forth in SEQ ID NO: 1, or amino acid residues corresponding to Lys 219 of human C1qA as set forth in SEQ ID NO: 1; and amino acid residue Ser 185 of human C1qC as set forth in SEQ ID NO: 3, or amino acid residues corresponding to Ser 185 of human C1qC as set forth in SEQ ID NO: 3.

[0270] In some embodiments, the humanized anti-C1q antibodies of the present disclosure can bind to the C1q protein and bind to one or more amino acids of the C1q protein at an amino acid residue selected from the group consisting of: (a) amino acid residues 218-240 of SEQ ID NO: 3 (SEQ ID NO: 20), or amino acid residues of C1qC corresponding to amino acid residues 218-240 of SEQ ID NO: 3 (WLAVNDYYDMVGIQGSDSVFSGF) (SEQ ID NO: 20); (b) amino acid residues 225-240 of SEQ ID NO: 3 (SEQ ID NO: 21), or amino acid residues of C1qC corresponding to amino acid residues 225-240 of SEQ ID NO: 3 (YDMVGI QGSDSVFSGF) (SEQ ID NO: 21); (c) amino acid residues 225-232 of SEQ ID NO: 3 (SEQ ID NO: 22), or amino acid residues of C1qC corresponding to amino acid residues 225-232 of SEQ ID NO: 3 (YDMVGIQG) (SEQ ID NO: (d) amino acid residue Tyr 225 of SEQ ID NO: 3, or an amino acid residue of C1qC corresponding to amino acid residue Tyr 225 of SEQ ID NO: 3; (e) amino acid residues 174-196 of SEQ ID NO: 3 (SEQ ID NO: 23), or an amino acid residue of C1qC corresponding to amino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT) of SEQ ID NO: 3 (SEQ ID NO: 23); (f) amino acid residues 184-192 of SEQ ID NO: 3 (SEQ ID NO: 24), or an amino acid residue of C1qC corresponding to amino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO: 3 (SEQ ID NO: 24); (g) amino acid residues 185-187 of SEQ ID NO: 3, or an amino acid residue of C1qC corresponding to amino acid residues 185-187 of SEQ ID NO: 3 (SEQ ID NO: 23); amino acid residues 185-187 (SGV) of C1qC of SEQ ID NO:3; (h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acid residue of C1qC corresponding to amino acid residue Ser 185 of SEQ ID NO:3.

[0271] anti-C1q Fab fragment

[0272] Before the advent of recombinant DNA technology, proteolytic enzymes (proteases) that cleave polypeptide sequences were used to dissect the structure of antibody molecules and determine which parts of the molecule are responsible for their various functions. Limited digestion with papain cleaves the antibody molecule into three fragments. Two of the 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 consisting of a V chain that is identical to the heavy chain. H and C H The Fab fragment is composed of a complete light chain with 1 domain pairing. Other fragments do not contain antigen binding activity, but were initially observed to be easily crystallized and therefore named as Fc fragments (crystallizable fragments). When Fab molecules were compared with IgG molecules, it was found that Fab was superior to IgG for certain in vivo applications because it has higher mobility and tissue penetration ability, its circulation half-life is shortened, it can monovalently bind antigen without mediating antibody effector functions, and its immunogenicity is lower.

[0273] Fab molecules are artificial fragments of approximately 50-kDa Ig molecules whose heavy chains are separated by the constant domain C H 2 and C H 3 shortened. Two heterophiles (V L -V H and C L -C H 1) Domain interaction is the basis of the double-chain structure of Fab molecules, which is achieved through C L with C H Fab and IgG have the same antigen binding site formed by six complementary determining regions (CDRs), V L and V H Three of each (LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3). CDRs define the highly variable antigen-binding site of an antibody. The highest sequence variation is found in LCDR3 and HCDR3, which are respectively represented by V and V in the natural immune system. L and J L Gene or V H 、D H and J H The rearrangement of genes produces LCDR3 and HCDR3. LCDR3 and HCDR3 usually form the core of the antigen binding site. The conserved region that connects and displays the six CDRs is called the framework region. In the three-dimensional structure of the variable domain, the framework region forms a sandwich of two antiparallel β-folds, which are connected by the outer hypervariable CDR loops and the inner conserved disulfide bonds. The unique combination of stability and versatility of the antigen binding sites of Fab and IgG is the basis of their success in clinical practice for disease diagnosis, monitoring, prevention and treatment.

[0274] All anti-Clq antibody Fab fragment sequences are incorporated by reference into US Patent Application No. 15 / 360,549, the antibodies and related compositions disclosed therein being incorporated herein by reference.

[0275] In certain embodiments, the present disclosure provides for binding to a protein comprising a heavy chain (V H / C H 1) and light chain (V L / C L ), wherein the anti-C1q antibody Fab fragment has six complementarity determining regions (CDRs), V L and V H Three of 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: 39) and comprises the following amino acid sequence:

[0276]

[0277] The complementarity determining regions (CDRs) of SEQ ID NO: 1 are in bold and underlined.

[0278] The light chain domain of the antibody Fab fragment comprises the following amino acid sequence (SEQ ID NO: 40):

[0279]

[0280] The complementarity determining regions (CDRs) of SEQ ID NO: 2 are in bold and underlined.

[0281] Anti-complement C1s antibody

[0282] Suitable inhibitors include antibodies that bind to the complement C1s protein (i.e., anti-complement C1s antibodies, also referred to herein as anti-C1s antibodies and C1s antibodies) and nucleic acid molecules encoding such antibodies. Complement C1s is an attractive target because it is located upstream in the complement cascade and has a narrow range of substrate specificity. In addition, it is possible to obtain antibodies (e.g., but not limited to, monoclonal antibodies) that specifically bind to the activated form of C1s.

[0283] All sequences mentioned in the following two paragraphs are incorporated by reference into U.S. Patent Application No. 14 / 890,811, the antibodies and related compositions disclosed therein being incorporated by reference herein.

[0284] In certain aspects, disclosed herein are methods of administering an anti-C1s antibody. The antibody can be a murine antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3 of the murine anti-human C1s monoclonal antibody 5A1, and the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3 of the murine anti-human C1s monoclonal antibody 5A1, produced by a hybridoma cell line deposited with the ATCC on May 15, 2013, or a progeny thereof (ATCC Accession No. PTA-120351). In other embodiments, the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3 of the mouse anti-human C1s monoclonal antibody 5C12, and the heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3 of the mouse anti-human C1s monoclonal antibody 5C12, produced by the hybridoma cell line deposited with the ATCC on May 15, 2013 (ATCC Accession No. PTA-120352), or a progeny thereof.

[0285] In some embodiments, the antibody specifically binds to and inhibits a biological activity of C1s or a C1s zymogen, such as the binding of C1s to C1q, the binding of C1s to C1r, or the binding of C1s to C2 or C4. The biological activity can be the proteolytic activity of C1s, the conversion of the C1s zymogen to an active protease, or the proteolytic cleavage of C2 or C4. In certain embodiments, the biological activity is activation of the classical complement activation pathway, activation of antibodies and complement-dependent cytotoxicity, or C1F hemolysis.

[0286] All sequences in the following sixty-two paragraphs are incorporated by reference into US Patent No. 8,877,197 to Van Vlasselaer, the disclosure of which antibodies and related compositions are incorporated herein by reference.

[0287] Disclosed herein are methods of administering a humanized monoclonal antibody that specifically binds to an epitope within a region encompassing domains IV and V of complement component C1s. In some instances, the antibody inhibits the binding of C1s to complement component 4 (C4) and / or does not inhibit the protease activity of C1s. In some embodiments, the method comprises administering a humanized monoclonal antibody that binds with high affinity to complement component C1s in the C1 complex.

[0288] Disclosed herein are methods of administering an anti-C1s antibody having one or more complementarity determining regions (CDRs) of an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and / or one or more CDRs of an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58. The anti-C1s antibody can bind to human or rat complement C1s protein. In some embodiments, the anti-C1s antibody inhibits the cleavage of at least one substrate cleaved by the complement C1s protein.

[0289] In certain embodiments, the antibody comprises: a) a complementarity determining region (CDR) having an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56; and / or b) a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0290] The antibody may comprise a CDR-L1 having an amino acid sequence of SEQ ID NO:51, a CDR-L2 having an amino acid sequence of SEQ ID NO:52, a CDR-L3 having an amino acid sequence of SEQ ID NO:53, a CDR-H1 having an amino acid sequence of SEQ ID NO:54, a CDR-H2 having an amino acid sequence of SEQ ID NO:55, and a CDR-H3 having an amino acid sequence of SEQ ID NO:56.

[0291] In other embodiments, the antibody may comprise a light chain CDR of a variable region having the amino acid sequence of SEQ ID NO:67 and / or a heavy chain CDR of a variable region having the amino acid sequence of SEQ ID NO:68.

[0292] The antibody can be a humanized antibody that specifically binds complement component C1s, wherein the antibody competes for epitope binding with an antibody comprising one or more CDRs of an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 67 and / or one or more CDRs of an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58 or SEQ ID NO: 68.

[0293] In other cases, the antibody can be a humanized antibody that specifically binds to complement C1s, wherein the antibody is selected from: a) a humanized antibody that specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for epitope binding with an antibody comprising a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; and b) a humanized antibody that specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for epitope binding with an antibody comprising a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 53, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. In some cases, the antibody competes for epitope binding with an antibody comprising heavy and light chain CDRs comprising: a) SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:69, SEQ ID NO:55, and SEQ ID NO:56; or b) SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0294] An antibody may comprise a light chain region and a heavy chain region present in a single polypeptide.An antibody may comprise an Fc region.

[0295] Disclosed herein are anti-C1s antibodies comprising a light chain variable region comprising an amino acid sequence 90% identical to SEQ ID NO:57, and a heavy chain variable region comprising an amino acid sequence 90% identical to SEQ ID NO:58.

[0296] The anti-C1s antibody can be selected from an antigen-binding fragment, an Ig monomer, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a scFv, a scAb, a dAb, a Fv, a single domain heavy chain antibody, a single domain light chain antibody, a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0297] Disclosed herein are methods of administering an antibody that competes for binding to an epitope bound by antibody IPN003 (also referred to herein as "IPN-M34" or "M34" or "TNT003"), e.g., an antibody comprising a variable domain of antibody IPN003, e.g., antibody IPN003.

[0298] In some embodiments, the method comprises administering an antibody that specifically binds to an epitope within the complement C1s protein. In some embodiments, the isolated anti-C1s antibody binds to an activated C1s protein. In some embodiments, the isolated anti-C1s antibody binds to an inactive form of C1s. In other cases, the isolated anti-C1s antibody binds to both the activated C1s protein and the inactivated form of C1s.

[0299] In some embodiments, the method comprises administering a monoclonal antibody that inhibits the cleavage of C4, wherein the isolated monoclonal antibody does not inhibit the cleavage of C2. In some embodiments, the method comprises administering a monoclonal antibody that inhibits the cleavage of C2, wherein the isolated monoclonal antibody does not inhibit the cleavage of C4. In some cases, the isolated monoclonal antibody is humanized. In some cases, the antibody inhibits a component of the classical complement pathway. In some cases, the component of the classical complement pathway inhibited by the antibody is C1s. The present disclosure also provides a method for treating a complement-mediated disease or condition by administering to an individual in need thereof an isolated monoclonal antibody that inhibits the cleavage of C4 or a pharmaceutical composition comprising the isolated monoclonal antibody, wherein the isolated monoclonal antibody does not inhibit the cleavage of C2.

[0300] In some embodiments, the method comprises administering a monoclonal antibody that inhibits C1s from cleaving C2 or C4, i.e., inhibiting C1s-mediated proteolytic cleavage of C2 or C4. In some cases, the monoclonal antibody is humanized. In some cases, the antibody inhibits C1s from cleaving C2 or C4 by inhibiting the binding of C2 or C4 to C1s; for example, in some cases, the antibody inhibits C1s-mediated cleavage of C2 or C4 by inhibiting the binding of C2 or C4 to the C2 or C4 binding site of C1s. Thus, in some cases, the antibody acts as a competitive inhibitor. The present disclosure also provides a method for treating epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy) by administering to an individual in need thereof an isolated monoclonal antibody that inhibits C1s from cleaving C2 or C4 (i.e., inhibiting C1s-mediated proteolytic cleavage of C2 or C4).

[0301] In some embodiments, the method comprises administering a monoclonal antibody that inhibits the cleavage of C4 by C1s, wherein the antibody does not inhibit the cleavage of complement component C2 by C1s; that is, the antibody inhibits C1s-mediated cleavage of C4, but does not inhibit C1s-mediated cleavage of C2. In some cases, the monoclonal antibody is humanized. In some cases, the monoclonal antibody inhibits the binding of C4 to C1s, but does not inhibit the binding of C2 to C1s. In some embodiments, the method comprises treating a complement-mediated disease or condition by administering to an individual in need thereof an isolated monoclonal antibody that inhibits the cleavage of C4 by C1s, wherein the antibody does not inhibit the cleavage of complement component C2 by C1s; that is, the antibody inhibits C1s-mediated cleavage of C4, but does not inhibit C1s-mediated cleavage of C2. In some embodiments of the method, the antibody is humanized.

[0302] In some embodiments, the method comprises administering a humanized monoclonal antibody that specifically binds to an epitope within the region encompassing domains IV and V of C1s. For example, the humanized monoclonal antibody specifically binds to an epitope within amino acids 272-422 of the amino acid sequence depicted in Figure 1 and set forth in SEQ ID NO: 70. In some cases, the humanized monoclonal antibody specifically binds to an epitope within amino acids 272-422 of the amino acid sequence depicted in Figure 1 and set forth in SEQ ID NO: 70 and inhibits binding of C4 to C1s. In some embodiments, the method comprises treating a complement-mediated disease or disorder by administering to an individual in need thereof a humanized monoclonal antibody that specifically binds to an epitope within amino acids 272-422 of the amino acid sequence depicted in Figure 1 and set forth in SEQ ID NO: 70 and inhibits binding of C4 to C1s.

[0303] In some embodiments, the method comprises administering a humanized monoclonal antibody that specifically binds to a conformational epitope within the region encompassing domains IV and V of C1s. For example, a humanized monoclonal antibody that specifically binds to a conformational epitope within amino acids 272-422 of the amino acid sequence depicted in Figure 1 and set forth in SEQ ID NO: 70. In some cases, the humanized monoclonal antibody specifically binds to a conformational epitope within amino acids 272-422 of the amino acid sequence depicted in Figure 1 and set forth in SEQ ID NO: 70 and inhibits binding of C4 to C1s. In some embodiments, the method comprises administering to an individual in need thereof a humanized monoclonal antibody that specifically binds to a conformational epitope within amino acids 272-422 of the amino acid sequence depicted in Figure 1 and set forth in SEQ ID NO: 70 and inhibits binding of C4 to C1s. In some embodiments, the method comprises administering to an individual in need thereof a humanized monoclonal antibody that specifically binds to a conformational epitope within amino acids 272-422 of the amino acid sequence depicted in Figure 1 and set forth in SEQ ID NO: 70 and inhibits binding of C4 to C1s.

[0304] In some embodiments, the method comprises administering a monoclonal antibody that binds to complement component C1s in the C1 complex. The C1 complex is composed of six C1q molecules, two C1r molecules, and two C1s molecules. In some cases, the monoclonal antibody is humanized. Thus, in some cases, the humanized monoclonal antibody that binds to complement component C1s in the C1 complex. In some cases, the antibody binds with high affinity to C1s present in the C1 complex.

[0305] In some embodiments, an anti-C1s antibody (e.g., a subject antibody that specifically binds an epitope in a complement C1s protein) comprises: a) a light chain region comprising one, two, or three VL CDRs of the IPN003 antibody; and b) a heavy chain region comprising one, two, or three VH CDRs of the IPN003 antibody; wherein the VH and VL CDRs are defined by Kabat (see, e.g., Table 1; and Kabat 1991).

[0306] In other embodiments, an anti-C1s antibody (e.g., a subject antibody that specifically binds an epitope in a complement C1s protein) comprises: a) a light chain region comprising one, two, or three VL CDRs of the IPN003 antibody; and b) a heavy chain region comprising one, two, or three VH CDRs of the IPN003 antibody; wherein the VH and VL CDRs are defined by Chothia (see, e.g., Table 1 and Chothia 1987).

[0307] Table 2 provides the CDR amino acid sequences and VL and VH amino acid sequences of the IPN003 antibody. Table 2 also provides the SEQ ID NO assigned to each amino acid sequence.

[0308] In some embodiments, an anti-C1s antibody (e.g., a subject antibody that specifically binds an epitope in a complement C1s protein) comprises: a) a light chain region comprising one, two, or three CDRs selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53; and b) a heavy chain region comprising one, two, or three CDRs selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56. In some embodiments, the anti-C1s antibody includes humanized VH and / or VL framework regions.

[0309] SEQ ID NO.51: SSVSSSYLHWYQ;

[0310] SEQ ID NO.52: STSNLASGVP;

[0311] SEQ ID NO.53: HQYYRLPPIT;

[0312] SEQ ID NO.54: GTFFSNYAMSWV;

[0313] SEQ ID NO.55: ISSGGSHTYY;

[0314] SEQ ID NO. 56: ARLFTGYAMDY.

[0315] In some embodiments, the anti-C1s antibody comprises a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0316] In some embodiments, an anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

[0317] In some embodiments, an anti-C1s antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0318] In some embodiments, the anti-C1s antibody comprises a CDR-L1 having an amino acid sequence of SEQ ID NO:51, a CDR-L2 having an amino acid sequence of SEQ ID NO:52, a CDR-L3 having an amino acid sequence of SEQ ID NO:53, a CDR-H1 having an amino acid sequence of SEQ ID NO:54, a CDR-H2 having an amino acid sequence of SEQ ID NO:55, and a CDR-H3 having an amino acid sequence of SEQ ID NO:56.

[0319] In some embodiments, an anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:57.

[0320] SEQ ID NO.57:

[0321] DIVMTQTTAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK.

[0322] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO. 58.

[0323] SEQ ID NO.58:

[0324] QVKLEESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATISSGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGYAMDYWGQGTSVT.

[0325] In some embodiments, an anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:57.

[0326] In some embodiments, an anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:58.

[0327] In some embodiments, an anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:57.

[0328] In some embodiments, an anti-C1s antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:58.

[0329] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:57 and a heavy chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:58.

[0330] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:57 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:58.

[0331] In some embodiments, the anti-C1s antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope with an antibody comprising a light chain CDR comprising the amino acid sequence of SEQ ID NO:57 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NO:58.

[0332] In some embodiments, the anti-C1s antibody comprises a light chain CDR comprising an antibody light chain variable region having the amino acid sequence of SEQ ID NO:57 and a heavy chain CDR comprising an antibody heavy chain variable region having the amino acid sequence of SEQ ID NO:58.

[0333] In some embodiments, an anti-C1s antibody (e.g., a subject antibody that specifically binds an epitope in a complement C1s protein) comprises: a) a light chain region comprising one, two, or three CDRs selected from SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:53; and b) a heavy chain region comprising one, two, or three CDRs selected from SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0334] SEQ ID NO.62: TASSSVSSSYLH;

[0335] SEQ ID NO.63: STSNLAS;

[0336] SEQ ID NO.53:HQYYRLPPIT;

[0337] SEQ ID NO.64: NYAMS;

[0338] SEQ ID NO.65: TISSGGSHTYYLDSVKG;

[0339] SEQ ID NO.66:LFTGYAMDY

[0340] In some embodiments, the anti-C1s antibody comprises a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0341] In some embodiments, an anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:53.

[0342] In some embodiments, an anti-C1s antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0343] In some embodiments, the anti-C1s antibody comprises a CDR-L1 having an amino acid sequence of SEQ ID NO:62, a CDR-L2 having an amino acid sequence of SEQ ID NO:63, a CDR-L3 having an amino acid sequence of SEQ ID NO:53, a CDR-H1 having an amino acid sequence of SEQ ID NO:64, a CDR-H2 having an amino acid sequence of SEQ ID NO:65, and a CDR-H3 having an amino acid sequence of SEQ ID NO:66.

[0344] In some embodiments, an anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:67.

[0345] SEQ ID NO.67:

[0346] QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK.

[0347] In some embodiments, an anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:68.

[0348] SEQ ID NO.68:

[0349] EVMLVESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATISSGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGYAMDYWGQGTSVTVSS.

[0350] In some embodiments, an anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:67.

[0351] In some embodiments, an anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:68.

[0352] In some embodiments, an anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:67.

[0353] In some embodiments, an anti-C1s antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:68.

[0354] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:67 and a heavy chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:68.

[0355] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 95% identical to the amino acid sequence of SEQ ID NO:67 and a heavy chain variable region comprising an amino acid sequence that is 95% identical to the amino acid sequence of SEQ ID NO:68.

[0356] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:67 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:68.

[0357] In some embodiments, the anti-C1s antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope with an antibody comprising a light chain CDR comprising the amino acid sequence of SEQ ID NO:67 and a heavy chain CDR comprising the amino acid sequence of SEQ ID NO:68.

[0358] In some embodiments, the anti-C1s antibody comprises a light chain CDR comprising an antibody light chain variable region having the amino acid sequence of SEQ ID NO:67 and a heavy chain CDR comprising an antibody heavy chain variable region having the amino acid sequence of SEQ ID NO:68.

[0359] In some embodiments, an anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:67.

[0360] In some embodiments, an anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:68.

[0361] An anti-C1s antibody may comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:79 and depicted in Figure 2 (VH variant 1).

[0362] An anti-C1s antibody may comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:80 and depicted in Figure 3 (VH variant 2).

[0363] An anti-C1s antibody may comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:81 and depicted in Figure 4 (VH variant 3).

[0364] An anti-C1s antibody may comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:82 and depicted in Figure 5 (VH variant 4).

[0365] An anti-C1s antibody may comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:83 and depicted in Figure 6 (VK variant 1).

[0366] An anti-C1s antibody may comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:84 and depicted in Figure 7 (VK variant 2).

[0367] An anti-C1s antibody may comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:85 and depicted in Figure 8 (VK variant 3).

[0368] An anti-C1s antibody may comprise a heavy chain variable region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 framework (FR) amino acid substitutions relative to the IPN003 parent antibody FR amino acid sequence, as shown in Table 3 ( Figure 9 ).

[0369] Complement inhibition

[0370] Many molecules that inhibit complement activity are known. In addition to known compounds, suitable inhibitors can be screened using the methods described herein. As described above, normal cells can produce proteins that block complement activity, such as CD59 and C1 inhibitors. In some embodiments of the present disclosure, complement is inhibited by upregulating the expression of genes encoding such polypeptides.

[0371] Modifications of molecules that block complement activation are also known in the art. For example, such molecules include, but are not limited to, modified complement receptors, such as soluble CR1. The mature protein of the most common CR1 allotype 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 consisting of 60 to 70 amino acid residues. Recent data indicate that C1q specifically binds to human CR1. Therefore, CR1 recognizes all three complement opsonins, namely C3b, C4b, and C1q. Soluble forms of recombinant human CR1 (sCR1) lacking the transmembrane and cytoplasmic domains have been produced and shown to retain all known functions of native CR1. The cardioprotective effect of sCR1 has been demonstrated in animal models of ischemia / reperfusion injury. Several types of human C1q receptors (C1qRs) have been described. These include a widely distributed 60- to 67-kDa receptor, termed cC1qR because it binds to the collagen-like domain of C1q. This C1qR variant has been shown to be a 126-kDa receptor for calreticulin, which regulates monocyte phagocytosis. gC1qR is not a membrane-bound molecule but rather a secreted soluble protein with affinity for the globular region of C1q and may serve as a fluid-phase regulator of complement activation.

[0372] Decay-accelerating factor (DAF) (CD55) consists of four SCRs plus a serine / threonine-rich domain that is capable of extensive O-linked glycosylation. DAF is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor and, through its ability to bind C4b and C3b, exerts its effects by dissociating C3 and C5 convertases. Soluble DAF (sDAF) has been shown to inhibit complement activation.

[0373] 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 modulates the classical pathway of complement activation by blocking the active sites of C1r and C1s and dissociating them from C1q.

[0374] Peptide inhibitors of complement activation include C5a, and other inhibitory molecules include fucoidan.

[0375] Nucleic acids, vectors and host cells

[0376] Antibodies suitable for use in the methods of the present disclosure can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In some embodiments, isolated nucleic acids having nucleotide sequences encoding any of the antibodies of the present disclosure are provided. Such nucleic acids can encode VL / C L and / or containing the amino acid sequence of an anti-C1q, anti-C1r or anti-C1s antibody H / C H 1 amino acid sequence. In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. Host cells containing such nucleic acids can also be provided. The host cells can contain the following (e.g., have been transduced with the following): (1) a V vector encoding an antibody; L / C L The amino acid sequence and V containing antibody H / C H 1 amino acid sequence of the nucleic acid vector, or (2) containing a V encoding an antibody containing L / C L The first vector of the nucleic acid containing the amino acid sequence of H / C H In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In some embodiments, the host cell is a bacterium, such as Escherichia coli.

[0377] Disclosed herein are methods for producing anti-C1q, anti-C1r, or anti-C1s antibodies. The methods comprise culturing a host cell of the present disclosure containing a nucleic acid encoding an anti-C1q, anti-C1r, or anti-C1s antibody under conditions suitable for antibody expression. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

[0378] To recombinantly produce the humanized anti-C1q, anti-C1r, or anti-C1s antibodies of the present disclosure, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily 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).

[0379] Suitable vectors containing the nucleic acid sequence encoding any antibody or its fragment polypeptide (including antibody) of the present disclosure 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 to be used, useful cloning vectors generally have the ability to self-replicate, can have a single target for a specific restriction endonuclease, and / or can carry genes for markers that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors can be obtained from commercial suppliers, such as BioRad, Stratagene, and Invitrogen.

[0380] Vectors containing nucleic acids of interest can be introduced into host cells by any of a number of suitable methods, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE dextran, or other substances; microparticle bombardment; lipid infection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of the vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell. In some embodiments, the vector comprises a nucleic acid comprising one or more amino acid sequences encoding an anti-C1q, anti-C1r, or anti-C1s antibody of the present disclosure.

[0381] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, the anti-C1q, anti-C1r, or anti-C1s antibodies of the present disclosure can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria (e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describe expression of antibody fragments in E. coli.). In other embodiments, the antibodies of the present disclosure can be produced in eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells) (e.g., U.S. Patent Application No. 14 / 269,950; U.S. Patent No. 8,981,071; Eur J Biochem. 1991 Jan 1; 195(1):235-42). After expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0382] Conditions of concern

[0383] Epilepsy is a group of neurological disorders in which nerve cell activity in the brain is disrupted, leading to seizures or periods of abnormal behavior, sensation, and sometimes loss of consciousness. Seizures can range from brief, barely noticeable fits to prolonged, intense shaking. In epilepsy, seizures tend to recur and have no immediate underlying cause. The cause of most cases of epilepsy is unknown, although some people develop epilepsy as a result of brain injury (such as TBI), stroke, brain tumors, and substance use disorders. Gene mutations are associated with a small number of diseases. Seizures are the result of excessive and abnormal activity of cortical nerve cells in the brain. Diagnosis typically involves ruling out other conditions that may cause similar symptoms, such as fainting. Furthermore, making a diagnosis involves determining whether there are any other causes of seizures, such as alcohol withdrawal or electrolyte problems. This can be done by imaging the brain and performing blood tests. Epilepsy can usually be confirmed with an electroencephalogram (EEG), but normal examinations cannot rule out the condition. Other brain imaging techniques, such as functional magnetic resonance imaging (fMRI), magnetic resonance spectroscopy (MRS), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), can also detect a form of epilepsy.

[0384] Epilepsy can be caused by many other conditions, including tumors, stroke, head trauma, previous infections of the central nervous system, genetic abnormalities, and brain damage before and after birth. There are several types of epilepsy, each with different causes, symptoms, and treatments. The two major types of epilepsy are idiopathic (genetic cause) and symptomatic or cryptogenic (presumed symptomatic, cause unknown). In idiopathic generalized epilepsy, there is usually (but not always) a family history of epilepsy. Idiopathic generalized epilepsy tends to appear in childhood or adolescence, although it may not be diagnosed until adulthood. In this type of epilepsy, apart from the seizures, no abnormalities of the nervous system (brain or spinal cord) are identified on EEG or imaging studies (MRI). On brain magnetic resonance imaging (MRI) scans, the brain is structurally normal, although certain studies may show scarring or subtle changes in the brain, which may have been present since birth. People with idiopathic generalized epilepsy have normal intelligence, and the results of the neurological examination and MRI are usually normal. Results of an electroencephalogram (EEG—a test that measures electrical impulses in the brain) can show epileptic discharges that affect a single area of ​​the brain or multiple areas (so-called generalized discharges). Types of seizures that affect people with idiopathic generalized epilepsy may include myoclonic seizures (sudden, very brief twitching of a limb), absence seizures (staring seizures), and / or generalized tonic-clonic seizures (grand mal seizures). Idiopathic generalized epilepsy is usually treated with medication. Some people outgrow the condition and stop having seizures, such as in cases of childhood absence epilepsy and a large number of people with juvenile myoclonic epilepsy.

[0385] Idiopathic partial epilepsy begins in childhood (between the ages of 5 and 8) and may be part of a family history. Also known as benign focal epilepsy of childhood (BFEC), this is considered one of the mildest types of epilepsy. It almost always disappears by adolescence and is never diagnosed in adults. Seizures tend to occur during sleep and are most commonly simple partial motor seizures involving the face and secondarily generalized (grand mal) seizures. This type of epilepsy is usually diagnosed using an electroencephalogram (EEG).

[0386] Symptomatic generalized epilepsy is caused by extensive brain damage. Injury during birth is the most common cause of symptomatic generalized epilepsy. In addition to seizures, these patients usually have other neurological problems, such as mental retardation or cerebral palsy. Specific hereditary encephalopathy, such as adrenoleukodystrophy (ADL) or brain infection (such as meningitis and encephalitis) also can cause symptomatic generalized epilepsy. When the cause of symptomatic generalized epilepsy can not be identified, this disease can be called cryptogenic epilepsy. These epilepsies include different subtypes, and the most common type is Lennox-Gastaut syndrome. Various types of seizures (generalized tonic-clonic seizures, tonic seizures, myoclonic seizures, tonic seizures, atonic seizures and absence seizures) are common in these patients and may be difficult to control.

[0387] Symptomatic partial (or focal) epilepsy is the most common type of epilepsy that begins in adults but also frequently occurs in children. This type of epilepsy is caused by a localized abnormality in the brain that can be caused by traumatic brain injury, stroke, tumors, trauma, congenital (present at birth) brain abnormalities, scarring or "sclerosis" of brain tissue, cysts, or infection. Sometimes, these brain abnormalities can be seen on MRI scans, but despite repeated attempts, they often cannot be identified because they are microscopic.

[0388] An example of a symptomatic partial (or focal) epilepsy is temporal lobe epilepsy (TLE), a group of conditions primarily involving amygdala-hippocampal dysfunction caused by neuronal hyperexcitability. Mesial temporal lobe epilepsy (MTLE), in particular, is perhaps the most electroclinically characterized of all epilepsy syndromes and the most common focal epilepsy in adults. At least 70% of patients with MTLE are resistant to currently available medications. The intrinsic potential of the temporal lobe to susceptibly develop focal seizures is based on a unique anatomical and functional network involving the amygdala-hippocampal complex and the entorhinal cortex. Most patients with refractory TLE exhibit severe unilateral hippocampal atrophy, so-called hippocampal sclerosis (HS), characterized histopathologically by segmental neuronal cell loss, astrogliosis, granule cell dispersion, and axonal reorganization in the CA1 and CA4 subfields. While the etiology of TLE is unknown in most cases (idiopathic), the condition is often associated with an initial, precipitating insult, including febrile seizures, trauma, stroke, brain infection, or status epilepticus (SE). It is generally agreed that such insults induce pathological changes in the brain that trigger the epileptogenic process and, after a latent period of months to years, lead to epilepsy. In addition to seizures, drug-resistant TLE is characterized by cognitive decline, particularly involving memory, accompanied by psychiatric co-morbidities. Behavioral deficits in TLE contribute significantly to the burden of the disease and often have a greater negative impact on patients' quality of life than the seizures themselves.

[0389] Pharmaceutical compositions and administration

[0390] The complement inhibitors (eg, antibodies) of the present disclosure can be administered in the form of pharmaceutical compositions.

[0391] Therapeutic formulations of the inhibitors (e.g., antibodies) of the present disclosure can be prepared by mixing the inhibitor having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions for storage (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed.

[1980] ). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used and include buffers (such as phosphates, acetates, 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 (less than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (such as polyvinyl pyrrolidone); amino acids (such as glycine, glutamine, asparagine, histidine, arginine, proline, and / or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins); chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (such as TWEEN®). TM 、PLURONICS TM or polyethylene glycol (PEG)).

[0392] Lipofection or liposomes can also be used to deliver antibodies or antibody fragments into cells, where an epitope or minimal fragment of the binding domain that specifically binds to the target protein is preferred.

[0393] The inhibitors can also be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980).

[0394] The formulations for administration may be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0395] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the inhibitor 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 polyvinyl alcohol), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT TM While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for shorter periods of time.

[0396] The antibodies and compositions of the present disclosure are typically administered by various routes, including but not limited to topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intralesional administration. Parenteral administration routes include intramuscular, intravenous, intraarterial, intraperitoneal, intravitreal, intrathecal, or subcutaneous administration.

[0397] The pharmaceutical composition may also contain (depending on the desired formulation) a pharmaceutically acceptable, non-toxic diluent carrier, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, normal 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 that approximate physiological conditions, such as pH adjusters and buffers, toxicity regulators, wetting agents, and detergents.

[0398] The composition can also include any of various stabilizers, such as antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide can be compounded with various well-known compounds that enhance the in vivo stability of the polypeptide, or enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance other pharmacokinetic and / or pharmacodynamic characteristics, or enhance solubility or absorption). Examples of such modifications or compounding agents include sulfates, gluconates, citrates, and phosphates. The polypeptide of the composition can also be compounded with molecules that enhance its in vivo properties. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids. Additional guidance on formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th edition (1985). For a brief review of the method for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0399] Toxicity and therapeutic efficacy of the active ingredients can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Therapeutic agents that exhibit high therapeutic indices are preferred.

[0400] The data obtained from cell culture and / or animal studies can be used to formulate a dosage range for humans. The dosage of the active ingredient is typically within a circulating concentration range that includes an ED50 with low toxicity. Dosage can vary within this range depending on the dosage form employed and the route of administration utilized.

[0401] The pharmaceutical compositions described herein can be administered in a variety of different ways. Examples include oral, intranasal, rectal, intravitreal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial administration of compositions containing a pharmaceutically acceptable carrier.

[0402] For oral administration, active ingredient can be used in solid dosage form, such as capsule, tablet and powder, or in liquid dosage form, such as elixir, syrup and suspension. One or more active ingredients can be encapsulated in gelatin capsule together with inactive ingredient and powdered carrier (such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivative, magnesium stearate, stearic acid, sodium saccharin, talcum powder, magnesium carbonate). The example of the extra inactive ingredient that can be added to provide the color, taste, stability, buffering capacity, dispersibility or other known desired features is red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide and edible white ink. Similar diluents can be used to prepare compressed tablets. Tablets and capsules can both be made into sustained-release products, to continuously release medicine in a few hours. Compressed tablets can be coated with sugar or film to cover any unpleasant taste and protect tablets from atmospheric influence, or enteric coating to selectively disintegrate in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring agents and flavorings to increase patient's acceptance.

[0403] Suitable formulations 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 include suspending agents, solubilizers, thickening agents, stabilizers and preservatives.

[0404] The components used to prepare the pharmaceutical composition are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, typically at least analytical grade, and more typically at least pharmaceutical grade). In addition, compositions intended for parenteral use are generally sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is generally free of any potential toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parenteral administration are also generally isotonic and are prepared under GMP conditions.

[0405] The compositions of the present disclosure can be administered using any medically appropriate procedure, for example, intravascular (intravenous, intraarterial, intracapillary), injection into the cerebrospinal fluid, intravitreal, topical, intracavitary or direct injection into the brain. Intrathecal administration can be performed using an Ommaya reservoir according to known techniques. (F. Balis et al., Am J. Pediatr. Hematol. Oncol. 11, 74, 76 (1989)).

[0406] When the therapeutic agent is locally applied in the brain, a method of administering the therapeutic composition of the present disclosure is to be deposited in or near the site by any suitable technique, such as by direct injection (if necessary, by means of the stereotactic positioning of an injection syringe) or by placing the tip of the Ommaya reservoir into a cavity or cyst for administration. Alternatively, a convection-enhanced delivery catheter can be directly implanted into the site, a natural or surgically produced cyst, or normal brain tissue. Such convection-enhanced pharmaceutical composition delivery devices greatly improve the diffusion of the composition throughout the brain tissue. The implanted catheters of these delivery devices utilize high-flow microinfusion (flow rate in the range of about 0.5 to 15.0 μl / minute), rather than diffusion flow, to deliver the therapeutic composition to the brain and / or tumor mass. Such devices are described in U.S. Patent number 5,720,720, which is incorporated herein by reference in its entirety.

[0407] The effective amount of a therapeutic composition administered to a particular patient will depend on various factors, some of which will vary from patient to patient. A competent clinician will be able to determine the effective amount of a therapeutic agent administered to a patient. The dosage of the agent will depend on the treatment, route of administration, therapeutic properties, patient sensitivity to the treatment, etc. Utilizing LD50 animal data and other information, clinicians can determine the maximum safe dose for an individual based on the route of administration. Utilizing common techniques, competent clinicians will be able to optimize the dosage of a particular therapeutic composition during routine clinical trials. The composition can be administered to a subject in a series of more than one administrations. For therapeutic compositions, it is sometimes necessary or desirable to administer them regularly. Treatment regimens vary depending on the agent; for example, some agents can be taken daily or half-day for a period of time, while more selective agents can be administered over a more defined time course, such as one, two, three or more days, one or more weeks, one or more months, etc., daily, half-day, half-weekly, weekly, etc.

[0408] The formulation can be optimized for retention and stability in the brain. When the agent is administered into the cranial cavity, it is desirable that the agent remain in the cavity and not diffuse or otherwise cross the blood-brain barrier. Stabilization techniques include cross-linking, polymerizing, or attaching to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc., to achieve an increase in molecular weight.

[0409] 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 biodegradability of the implant. The transport of the drug through the polymer barrier will also be affected by the solubility of the compound, the hydrophilicity of the polymer, the degree of polymer cross-linking, the swelling of the polymer to make the polymer barrier more permeable to the drug, the geometric shape of the implant, etc. The size of the implant is suitable for the size and shape of the area selected as the implant site. The implant can be a particle, a thin sheet, a patch, a plaque, a fiber, a microcapsule, etc., and can be any size or shape compatible with the selected insertion site.

[0410] Implants can be monolithic, i.e., the active agent is 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 used will vary with the site of administration, the desired duration of treatment, patient tolerance, the nature of the disease to be treated, etc. Characteristics of the polymer include biodegradability at the implant site, compatibility with the agent of interest, ease of encapsulation, and half-life in a physiological environment.

[0411] Biodegradable polymer compositions that can be used can be organic esters or ethers that produce physiologically acceptable degradation products upon degradation, including monomers. Anhydrides, amides, orthoesters, or the like, can be used by themselves or in combination with other monomers. The polymer can be a condensation polymer. The polymer can be cross-linked or non-cross-linked. Of particular interest are polymers (homopolymers or copolymers) of hydroxyaliphatic carboxylic acids and polysaccharides. Polyesters of interest include 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 biodegradable polymers are obtained, while racemates significantly enhance degradation. Copolymers of glycolic acid and lactic acid are of particular interest, where the biodegradation rate is controlled by the ratio of glycolic acid to lactic acid. The fastest-degrading copolymers contain approximately equal amounts of glycolic acid and lactic acid, with either homopolymer being more resistant to degradation. The ratio of glycolic acid to lactic acid will also affect the brittleness of the implant, with softer implants being desirable for larger geometries. Polysaccharides of interest include calcium alginate and functionalized cellulose, particularly carboxymethyl cellulose esters, which are characterized by being water-insoluble and having a molecular weight of about 5 kD to 500 kD. Biodegradable hydrogels can also be used in implants of the present invention. Hydrogels are typically copolymer materials characterized by the ability to absorb liquids. Exemplary biodegradable hydrogels that can be used are described in Heller, Hydrogels in Medicine and Pharmacy, compiled by N.A. Peppes, Vol. 3, CRC Press, Boca Raton, Fla., 1987, pp. 137-149.

[0412] Treatment

[0413] The methods of the present invention modulate the immune response to epilepsy by administering complement inhibitors. Epilepsy can be induced by traumatic brain injury (TBI), anoxic brain injury, brain infection, stroke, or genetic syndromes. In a preferred embodiment, the epilepsy is TBI-induced epilepsy. Without being bound by theory, immature astrocytes induce the expression of C1q protein in neurons during development. In patients with TLE, there is evidence of microglial activation in the hippocampus, suggesting that the immune response is activated. Inflammatory mediators such as complement factors are normally expressed at very low levels in healthy brain tissue, but can be rapidly induced by various brain insults such as infection, ischemia, injury, and seizures. Activation of C1q, C1r, and C1s contributes to the inflammatory response, leading to synaptic loss, as well as the generation and recurrence of seizures and seizure-related neuronal damage. Unregulated persistent inflammation, blood-brain barrier damage, and uncontrolled seizures trigger the progression of TLE. During the development of TLE, overexpression of C1q, C1r, and C1s can be coupled with complement activation signals (eg, β-amyloid, APP, cytokines (such as IFNγ, TNFα), etc.), and also lead to inflammation.

[0414] By administering drugs that inhibit complement activation, synapses that would otherwise be lost can be preserved. Such agents include C1q, C1r, and C1s 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.

[0415] By administering agents that inhibit complement activation, the production of antigen-specific antibodies that mediate epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy) can be reduced. Such agents include anti-C1q, anti-C1r, or anti-C1s antibody inhibitors. Other agents may include inhibitors that upregulate the expression of natural complement, or agents that downregulate the synthesis of C1q, C1r, or C1s in cells, agents that block complement activation, agents that block complement activation signals, and the like.

[0416] The methods promote improved maintenance of neuronal function in conditions associated with synaptic loss. Maintenance of neural connections provides functional improvement in neurodegenerative diseases relative to untreated patients. Prevention of synaptic loss can include at least a measurable improvement in synapse number, such as at least a 10% improvement, at least a 20% improvement, at least a 50% improvement, or more, over a period of 1, 2, 3, 4, 5, 6 days, or at least a week relative to a control group lacking such treatment.

[0417] Preferably, the medicament of the present invention is administered at a dose that reduces synaptic loss while minimizing any side effects. It is contemplated that compositions for in vivo use will be obtained and used under the guidance of a physician. The dosage of the therapeutic formulation will vary widely depending on the nature of the disease, the frequency of administration, the mode of administration, the clearance of the drug from the host, and the like.

[0418] 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 techniques, a competent clinician will be able to adjust the dosage of a particular therapeutic agent or imaging composition in the course of routine clinical trials.

[0419] Therapeutic agents (e.g., complement inhibitors, gene expression activators, etc.) can be incorporated into various formulations for therapeutic administration by combining with appropriate pharmaceutically acceptable carriers or diluents, and can be formulated into solid, semisolid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Thus, administration of the compound can be achieved in various ways, including oral, oral, rectal, parenteral, intraperitoneal, intradermal, transdermal, intrathecal, intranasal, intratracheal administration, etc. The active agent can be systemic after administration or can be localized by using regional administration, internal administration, or using an implant that acts to maintain the active dose at the implantation site.

[0420] One strategy for delivering drugs across the blood-brain barrier (BBB) ​​requires destroying the BBB, either by osmotic means such as mannitol or leukotrienes, or by biochemical means using vasoactive substances such as bradykinin. The possibility of using BBB opening to target specific drugs is also an option. When the composition is administered by intravascular injection, BBB disruptors can be co-administered with the therapeutic composition of the present invention. Other strategies for passing through the BBB may require the use of endogenous transport systems (including carrier-mediated transport proteins (such as glucose and amino acid carriers)), receptor-mediated transport endocytosis of insulin or transferrin, and active efflux transport proteins (such as p-glycoprotein). Active transport moieties can also be combined with therapeutic or imaging compounds for use in the present invention to promote transport across the vascular epithelial wall. Alternatively, drug delivery behind the BBB is by direct intrathecal delivery of therapeutic agents or imaging agents to the skull, such as through the Ommaya reservoir.

[0421] Method for neutralizing complement bioactivity. The complement bioactivity affected can be (1) binding of C1q to autoantibodies, (2) binding of C1q to C1r, (3) binding of C1q to C1s, (4) binding of C1q to IgM, (5) binding of C1q to IgG, (6) binding of C1q to phosphatidylserine, (7) binding of C1q to pentraxin-3, (8) binding of C1q to C-reactive protein (CRP), (9) binding of C1q to globular C1q receptor (gC1qR), (10) binding of C1q to complement receptor 1 (CR1), (11) binding of C1q to beta-amyloid, (12) binding of C1q to calreticulin, (13) binding of C1q to apoptotic cells, or (14) binding of C1q to B cells. The affected complement bioactivities can further be (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, and (12) activation of complement receptor 3 (CR3 / C3) expressing cells.

[0422] It is expected that the composition for in vivo use can be obtained and used under the guidance of a physician. The dosage of the therapeutic formulation can vary widely depending on the nature of the disease, the frequency of administration, the mode of administration, the clearance of the drug from the host body, etc.

[0423] The effective amount of a therapeutic composition administered to a particular patient will depend on a variety of factors, several of which will vary from patient to patient. Using ordinary techniques, a competent clinician will be able to adjust the dosage of a particular therapeutic agent or imaging composition in the course of routine clinical trials.

[0424] Therapeutic agents (e.g., complement inhibitors, gene expression activators, etc.) can be incorporated into various formulations for therapeutic administration by combining with appropriate pharmaceutically acceptable carriers or diluents, and can be formulated into solid, semisolid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Thus, administration of the compound can be achieved in various ways, including oral, oral, rectal, parenteral, intraperitoneal, intradermal, transdermal, intrathecal, intranasal, intratracheal administration, etc. The active agent can be systemic after administration or can be localized by using regional administration, internal administration, or using an implant that acts to maintain the active dose at the implantation site.

[0425] Compound screening

[0426] In one aspect of the invention, candidate drugs for use as inhibitors with the ability to modulate synaptic loss are screened. This compound screening can be performed using in vitro models, genetically modified cells or animals, or purified proteins. For this purpose, a variety of assays can be used. In one embodiment, compounds predicted to be complement antagonists or agonists (including specific complement proteins (e.g., C1q) and complement activation signals (e.g., beta-amyloid, APP), etc.) are tested in an in vitro culture system, as described below.

[0427] For example, candidate drugs can be identified by known pharmacology, by structural analysis, by rational drug design using computer-based modeling, by binding assays, and the like. Various in vitro models can be used to determine whether a compound binds to or otherwise affects complement activity. Such candidate compounds are used to contact neurons in an environment that allows for synaptic loss. Such compounds can be further tested for their effect on synaptic loss in in vivo models.

[0428] Screening can also be performed for molecules produced by astrocytes, such as immature astrocytes, that induce C1q expression in neurons. In such assays, a co-culture of neurons and astrocytes is assessed for the production or expression of molecules that induce C1q expression. For example, a blocking antibody can be added to the culture to determine the effect of induction of C1q expression in neurons.

[0429] Synapse loss is quantified by administering a candidate agent to neurons in culture and determining the presence of synapses in the absence or presence of the agent. In one embodiment of the invention, the neurons are primary cultures of, for example, RGCs. Purified populations of RGCs are obtained by conventional methods, such as sequential immunoselection. The cells are cultured in a suitable culture medium, which typically contains appropriate growth factors, such as CNTF; BDNF; etc. Neural cells (e.g., RCGs) are cultured for a period of time sufficient to allow robust process growth and then cultured with candidate agents for a period of about 1 day to 1 week. In many embodiments, neurons are cultured on a live astrocyte feeder layer to induce signaling of synaptic loss. Methods for culturing astrocyte feeder layers are known in the art. For example, cortical glial cells can be plated in a culture medium that does not allow neurons to survive, removing non-adherent cells.

[0430] For synaptic quantification, cultures are fixed, blocked, and washed, then stained with antibodies specific for synaptic proteins (e.g., synaptotagmin, etc.) and visualized with appropriate reagents known in the art. Staining analysis can be performed under a microscope. In one embodiment, a digital image of the fluorescence emission is acquired using a camera and image capture software, adjusted to remove the unused portion of the pixel value range, and the used pixel values ​​are adjusted to utilize the entire pixel value range. The corresponding channel images can be merged to create a color (RGB) image containing two single-channel images as separate color channels. A rolling ball background subtraction algorithm can be used to remove low-frequency background from each image channel to identify colocalized points. The number, average area, average minimum and maximum pixel intensity, and average pixel intensity of all synaptotagmin, PSD-95, and colocalized points in the image are recorded and saved to disk for analysis.

[0431] Drug candidates can be obtained from a variety of sources, including synthetic or natural compound libraries. For example, many methods can be used for the random and directed synthesis of various organic compounds and biomolecules, including the expression of random oligonucleotides and oligopeptides. Alternatively, natural compound libraries in the form of bacteria, fungi, plants, and animal extracts are available or easily produced. In addition, natural or synthetically produced libraries and compounds are easy to modify by conventional chemical, physical, and biochemical methods and can be used to produce combinatorial libraries. Known drugs can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs. Test reagents can be obtained from libraries, such as natural product libraries or combinatorial libraries.

[0432] Libraries of candidate compounds can also be prepared by rational design (see generally Cho et al., Pac. Symp. Biocompat. 305-16, 1998); Sun et al., J. Comput. Aided Mol. Des. 12:597-604, 1998); each of which is incorporated by reference in its entirety). For example, libraries of phosphatase inhibitors can be prepared by synthesis of combinatorial chemical libraries (see generally DeWitt et al., Proc. Nat. Acad. Sci. USA 90:6909-13, 1993; International Patent Publication WO 94 / 08051; Baum, Chem. & Eng. News, 72:20-25, 1994; Burbaum et al., Proc. Nat. Acad. Sci. USA 92:6027-31, 1995; Baldwin et al., J. Am. Chem. Soc. 117:5588-89, 1995; Nestler et al., J. Org. Chem. 59:4723-24, 1994; Borehardt et al., J. Am. Chem. Soc. 116:373-74, 1994; Ohlmeyer et al., Proc. Nat. Acad. Sci. USA 90:10922-26, all of which are incorporated herein by reference in their entirety).

[0433] Compounds initially identified by any screening method can be further tested to confirm apparent activity. The basic form of such an approach involves administering the lead compound identified in the initial screen to an animal model for humans and then determining the effect on synaptic loss. The animal model used in the validation study is typically a mammal. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats.

[0434] Combination therapy

[0435] The complement inhibitors of the present disclosure may be used, without limitation, in combination with any additional treatment for epilepsy, such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy.

[0436] In some embodiments, the antibodies, antibody fragments, and / or antibody derivatives of the present disclosure are administered in combination with a second inhibitory anti-complement factor antibody, such as an anti-C1q or anti-C1r antibody or an anti-C1s antibody. In some embodiments, the antibodies are administered together with a second and a third inhibitory anti-complement factor antibody, such as an anti-C1s antibody, an anti-C1q antibody, and / or an anti-C1r antibody.

[0437] In some embodiments, an inhibitor of the present disclosure is administered in combination with an inhibitor of antibody-dependent cellular cytotoxicity (ADCC). ADCC inhibitors may include, but are not limited to, soluble NK cell inhibitory receptors, such as killer cell Ig-like receptors (KIRs) (which recognize HLA-A, HLA-B, or HLA-C) and the C-type lectin CD94 / NKG2A heterodimer (which recognizes HLA-E) (see, e.g., López-Botet M., T. Bellón, M. Llano, F. Navarro, P. García & M. de Miguel. (2000), Paired inhibitory and triggering NK cell receptors for HLA class I molecules. Hum. Immunol. 61:7-17; Lanier LL (1998) Follow the leader: NK cell receptors for classical and nonclassical MHC class I. Cell 92:705-707.), and cadmium (see, e.g., Immunopharmacology 1990; Vol. 20, pp. 73-8).

[0438] In some embodiments, the antibodies, antibody fragments, and / or antibody derivatives 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; CD59, DAF, CR1, CR2, Crry, or compstatin-like peptides in the form of soluble, membrane-bound, labeled, or fusion proteins that block C3 cleavage; non-peptide C3aR antagonists such as SB 290157; cobra venom factor; or non-specific complement inhibitors such as nafamostat mesilate (FUTHAN; FUT-175); aprotinin; K-76 monocarboxylic acid (MX-1); and heparin (see, e.g., T. Mollnes & M. Kirschfink, Molecular Immunology 43 (2006) 107–121).

[0439] In some embodiments, the antibodies, antibody fragments, and / or antibody derivatives of the present disclosure are administered in combination with inhibitors of the interaction between autoantibodies and their autoantigens. Such inhibitors may include purified soluble forms of autoantigens, or antigen mimetics, such as peptide or RNA-derived mimotopes, including mimotopes of the AQP4 antigen. Alternatively, such inhibitors may include blocking agents that recognize autoantigens and prevent autoantibody binding without triggering the classical complement pathway. Such blocking agents may include, for example, autoantigen-binding RNA aptamers or antibodies that lack a functional C1q binding site in their Fc domain (e.g., Fab fragments or antibodies otherwise engineered not to bind C1q).

[0440] Reagent test kit

[0441] The present invention also provides kits containing the antibodies, antibody fragments and / or antibody derivatives of the present invention. The kits of the present invention include one or more containers containing the purified anti-C1s, anti-C1q or anti-C1r antibodies of the present invention and instructions for use according to methods known in the art. Generally, these instructions include a description of administering the inhibitor to treat or diagnose epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy or symptomatic partial epilepsy). The kit may further include a description of selecting an individual suitable for treatment based on the identification of whether the individual has the disease and the stage of the disease.

[0442] The instructions typically include information about dosage, dosing regimen, and route of administration for the intended treatment. The container can be a unit dose, bulk package (e.g., multi-dose package), or 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 magnetic or optical storage disk) are also acceptable.

[0443] The label or package insert may indicate that the composition is used to treat TBI-induced epilepsy.Instructions for use in treating TBI-induced epilepsy can be provided to practice any of the methods described herein.

[0444] The label or package insert may indicate that the composition is used to treat TLE.TLE instructions may be provided to practice any of the methods described herein.

[0445] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Also contemplated are packaging used in conjunction with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices (such as mini-pumps). The kit can have a sterile access port (e.g., the container can be an intravenous fluid bag or a vial with a stopper pierced by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous fluid bag or a vial with a stopper pierced by a hypodermic needle). At least one active agent in the composition is an inhibitor of the classical complement pathway. The container can further comprise a second pharmaceutically active agent.

[0446] The kit may optionally provide additional components, such as buffers and interpretative information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0447] Diagnostic uses

[0448] Although some people who have seizures have abnormal EEGs, many do not. Several additional tests are available to help identify the type of seizure and its effects. These include a complete neurology consultation for epilepsy and related conditions, neurophysiological testing including routine EEG, outpatient and inpatient video EEG monitoring, long-term inpatient video EEG monitoring using scalp or intracranial electrodes, neuroimaging (e.g., MRI, MRS, PET, fMRI), neuropsychology, and speech and auditory processing assessments.

[0449] For example, temporal lobe epilepsy is the most common form of partial or localized epilepsy. There are generally two types of temporal lobe epilepsy: one involving the medial or internal structures of the temporal lobe, and the second, called neocortical temporal lobe epilepsy, involving the outer part of the temporal lobe. Understanding several characteristics that can be helpful in determining a subject's risk of developing temporal lobe epilepsy (TLE) is important. A hallmark of TLE is simple focal seizures without loss of consciousness (with or without aura) or focal cognitive impairment seizures (with loss of consciousness). Loss of consciousness occurs during a focal cognitive impairment seizure, when the seizure spreads to both temporal lobes. In epidemiological terms, focal epilepsy typically originates in the temporal lobe, but the true prevalence of TLE is unknown. Regarding clinical manifestations, auras are present in most cases of temporal lobe epilepsy. Most auras and involuntary movements last very briefly—a few seconds or one to two minutes. Auras can cause sensory, autonomic, or psychiatric symptoms. Somatic and special sensory phenomena include olfactory, auditory, and gustatory illusions, as well as hallucinations. Patients may report distortions in the shape, size, and distance of objects. These visual errors differ from the visual hallucinations associated with occipital lobe attacks because no visual image is formed. For example, objects may appear smaller or larger than usual. In addition, vertigo may occur during attacks in the posterior superior temporal gyrus. Psychological phenomena include feelings of déjà vu (familiar) or newness (unfamiliarity), depersonalization (e.g., a feeling of being detached from oneself) or derealization (surroundings seem unreal), fear or anxiety, and patients may describe seeing their bodies from outside. Autonomic phenomena include changes in heart rate and sweating. Patients may experience a feeling of fullness in the upper abdomen or nausea.

[0450] After an aura, a temporal focal cognitive impairment episode begins with wide-open eyes, a motionless stare, dilated pupils, and cessation of behavior. Lip smacking, chewing, and swallowing may be noted. Involuntary hand movements or unilateral dystonic posturing may also occur. A focal cognitive impairment episode may evolve into a generalized tonic-clonic (GTC) seizure. Patients typically experience a postictal period of confusion. The postictal phase may last several minutes. Amnesia occurs during a focal cognitive impairment episode due to bilateral hemispheric involvement.

[0451] Possible underlying causes of TLE include previous infection (eg, herpes encephalitis or bacterial meningitis), traumatic brain injury, head injury producing a contusion or hemorrhage resulting in encephalomalacia or cortical scarring, anoxic brain injury, brain infection, stroke, hamartomas, gliomas, genetic syndromes, vascular malformations (eg, arteriovenous malformations, cavernous hemangiomas), cryptogenic (the cause is presumed but not yet determined), or idiopathic. Other potential causes of TLE include hippocampal sclerosis due to mesial temporal lobe epilepsy, which begins in late childhood, then remits, but reappears in a refractory form in adolescence or early adulthood. In addition, febrile seizures may precipitate TLE, as some children with complex febrile seizures appear to be at risk for developing TLE later in life.

[0452] Differential diagnosis is sometimes used to diagnose and evaluate individuals at risk for TLE. Some features of the differential diagnosis used in the diagnosis of TLE include excessive daytime sleepiness (e.g., due to sleep apnea or narcolepsy), periodic limb movement disorder, tardive dyskinesia, and occipital lobe epilepsy (which can extend to the temporal lobe and be clinically indistinguishable from temporal lobe seizures). Psychogenic seizures, meaning that patients with psychogenic seizures may also have epileptic seizures, are also used in the differential diagnosis of TLE. Absence seizures are characterized by a sudden onset without aura, typically lasting less than 30 seconds, without a postictal period of confusion, and without complex involuntary movements. Focal incitatory seizures are often preceded by a distinct aura, lasting more than a minute, and followed by a postictal period of confusion. Frontal lobe focal incitatory seizures present as brief episodes with abrupt onset and abrupt end. A very brief postictal state can occur, which may result in behavioral changes such as vocalizations and complex and sexual movements. Electroencephalography (EEG) mapping may be required to differentiate from TLE.

[0453] Additionally, regarding diagnostic and traditional methods for determining a subject's risk for temporal lobe epilepsy, MRI is often the neuroimaging diagnostic of choice. Conventional brain MRI using certain markers will detect lesions (e.g., small tumors, vascular malformations, and cortical dysplasia) that are not detected by computed tomography (CT). For example, one detectable marker is interictal [ 18 F]fluorodeoxyglucose-positron emission tomography ( 18 FDG-PET) has a sensitivity of 60%–90%. Another detectable label is arterial spin labeling (ASL), which can quantify regional cerebral blood flow by measuring the influx of magnetically labeled arterial blood into a target area.

[0454] MRI performed for the evaluation of drug-resistant epilepsy requires specialized protocols. For example, hippocampal sclerosis is characterized by neuronal loss and gliosis. HS is the most common pathological basis for surgical treatment of adult epilepsy and is seen in 67% of patients. HS has been reported in 1.5%-3% of adults with newly diagnosed epilepsy. When evaluating the medial temporal lobe structures (hippocampus, amygdala, entorhinal cortex, and hippocampal gyrus), MRI is used to assess size, signal, shape, and dual pathology (SSSD). The typical MRI findings of HS include hippocampal atrophy on T1-weighted SPGR (usually seen in 90%-95% of cases). Atrophy is most prominent in the hippocampus.

[0455] Using fluid-attenuated inversion recovery (FLAIR) imaging, an increased signal was observed in the hippocampus. FLAIR is well-suited to detecting signal changes in the hippocampus because changes in glial cells increase water content, which manifests as an increased signal on T2-weighted MRI. The FLAIR sequence eliminates the increased signal intensity of cerebrospinal fluid (CSF) in the temporal horns of the lateral ventricles and the choroidal fissure, which can dwarf the increased signal in the hippocampus on conventional thin-section T2-weighted spin-echo images. The baseline signal in the hippocampus on FLAIR MRI is greater than that in the cortex. In children, HS is observed in 21% of patients with newly diagnosed TLE and in up to 57% of patients with refractory TLE. More common findings in children with refractory TLE include MCD and developing tumors.

[0456] CT scans have a role in the emergency evaluation of seizures or when MRI is contraindicated (for example, when the patient has a pacemaker or metal implant). Noncontrast CT scans will not identify some vascular lesions and tumors. CT has a limited role in the evaluation of refractory epilepsy. Electrocardiography (ECG) can also be used to evaluate all patients with altered consciousness, especially older patients, in whom arrhythmias may mimic epilepsy. Twenty-four-hour ambulatory ECG and other cardiovascular tests (including those with implantable ring devices) may also be helpful. When MRI results are normal, the radioisotope fluorodeoxyglucose (18 F Positron emission tomography (PET) with FDG-PET as a detectable marker is useful. Interictal EEG is also used to obtain recordings from scalp electrodes, as one-third of patients with TLE have bilateral, independent, transient interictal epileptiform abnormalities. In addition, single-photon emission computed tomography (SPECT) is useful for candidates for surgical intervention, and video EEG telemetry is used as part of the preoperative evaluation. This approach can also be used if the diagnosis of TLE remains uncertain.

[0457] The present invention provides, in part, a method for determining a subject's risk of developing an epilepsy, such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy, the method comprising: administering an anti-C1q, anti-C1r, or anti-C1s antibody to the subject, wherein the anti-C1q, anti-C1r, or anti-C1s antibody is conjugated to a detectable label; detecting the detectable label to measure the amount or location of C1q, C1r, or C1s in the subject; and comparing the amount or location of one or more of C1q, C1r, or C1s to a reference, wherein the risk of developing an epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy) is characterized based on the comparison of the amount or location of one or more of C1q, C1r, or C1s to the reference.

[0458] An exemplary method for detecting the level of C1q, C1r, or C1s and therefore for classifying whether a sample is associated with epilepsy (such as idiopathic generalized epilepsy, idiopathic partial epilepsy, symptomatic generalized epilepsy, or symptomatic partial epilepsy, or a clinical subtype thereof) involves obtaining a biological sample from a test subject and contacting the biological sample with an antibody capable of detecting C1q, C1r, or C1s, thereby detecting the level of C1q, C1r, or C1s in the biological sample. In some cases, the statistical algorithm is a single learning statistical classification system. For example, the single learning statistical classification system can be used to classify a sample as a C1q, C1r, or C1s sample based on a predicted or probability value and the presence or level of C1q, C1r, or C1s. Use of a single learned statistical classification system typically classifies samples as C1q, C1r, or C1s samples with a sensitivity, specificity, positive predictive value, negative predictive value, and / or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0459] Other suitable statistical algorithms are well known to those skilled in the art. For example, learning statistical classification systems include machine learning algorithm technology that can adapt to complex data sets (e.g., groups of markers of interest) and make decisions based on such data sets. In some embodiments, a single learning statistical classification system is used, such as a classification tree (e.g., random forest). In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more learning statistical classification systems is used, preferably used in series. Examples of learning statistical classification systems include, but are not limited to, systems using inductive learning (e.g., decision trees / classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), probabilistic approximately correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro-fuzzy networks (NFN), network structures, perceptrons such as multilayer perceptrons, multilayer feedforward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in known environments such as naive learning, adaptive dynamic learning and temporal difference learning, passive learning in unknown environments, active learning in unknown environments, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classification systems include support vector machines (e.g., kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithm, Gauss-Newton algorithm, mixture Gaussian models, gradient descent algorithm, and learning vector quantization (LVQ).

[0460] In one embodiment, the method further involves obtaining a control biological sample (e.g., a biological sample from a subject not suffering from a condition or disorder mediated by C1q, C1r, or C1s), a biological sample from a subject during remission or prior to developing a condition or disorder mediated by C1q, C1r, or C1s, or a biological sample from a subject during treatment for developing a condition or disorder mediated by C1q, C1r, or C1s.

[0461] An exemplary method for detecting the presence or absence of C1q, C1r, or C1s is to administer an anti-C1q, anti-C1r, or anti-C1s antibody to a subject, wherein the anti-C1q, anti-C1r, or anti-C1s antibody is conjugated to a detectable label. In some embodiments, the detectable label comprises a nucleic acid, an oligonucleotide, an enzyme, a radioisotope, biotin, or a fluorescent label. In some embodiments, the detectable label is detected using an imaging agent for x-ray, CT, MRI, ultrasound, PET, and SPECT. In some embodiments, the fluorescent label is selected from fluorescein, rhodamine, a cyanine dye, or BODIPY.

[0462] It should be understood that one, some or all of the features of the various embodiments described herein can be combined to form other embodiments of the compositions and methods provided herein. All combinations of embodiments of the present invention are expressly included in the present invention and disclosed herein, just as each and every combination is individually and clearly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly included in the present invention and disclosed herein, just as each and every such subcombination is individually and clearly disclosed herein. These and other aspects of the compositions and methods provided herein will become apparent to those skilled in the art.

[0463] Example

[0464] Example 1: Materials and Methods

[0465] animal

[0466] Adult male CD1 mice (P30-P180) were used for most experiments. Adult male Thy1-GCaMP6f mice (Tg(Thy1-GCaMP6f)GP5.17Dkim ISMR_JAX:025393; C57BL / 6 congenic strain), wild-type C57BL / 6 mice (ISMR_JAX:000664), and C1q knockout mice (C1qatm1Mjw, ISMR_APB:1494; C57BL / 6 congenic strain) were used for specific experiments.

[0467] Controlled Cortical Impact (CCI)

[0468] Mice were anesthetized with 2-5% isoflurane and placed in a stereotaxic frame. A 3 mm craniotomy was performed on the right somatosensory cortex (S1) centered at -1 mm posterior to bregma and +3 mm lateral to the midline. TBI was performed using a CCI device equipped with a metal piston (CCI's Impact One stereotaxic impactor, Leica Microsystems) using the following parameters: 3 mm tip diameter, 15 ° angle, 0.8 mm depth from the dura mater, 3 m / s speed, and 100 ms dwell time. Sham-operated animals received the same anesthesia and craniotomy, but no injury was caused.

[0469] Immunostaining and microscopy

[0470] We anesthetized mice with a lethal dose of Fatal-Plus (see www.drugs.com / vet / fatal-plus-solution.html) and perfused with 4% paraformaldehyde in 1X PBS. Serial coronal sections (30 μm thick) were cut on a Leica SM2000R sliding microtome. Sections were incubated with antibodies against C1q (1:700, rabbit, Abcam, ab182451, 640AB_2732849), GFAP (1:1000, chicken, Abcam, ab4674, AB_304558), GFP (1:500, chicken, Aves Labs, AB_10000240), Iba1 (1:500, rabbit, Wako, 019-19741, AB_839504), and NeuN (1:500, mouse, Millipore, MAB377, AB_2298772) at 4°C. After washing, sections were incubated with Alexa Fluor-conjugated secondary antibodies (1:300, Thermo Fisher Scientific, A-11029) for two hours at room temperature. We mounted sections in antifade medium (Vectashield) and imaged them at 10–20× using a Biorevo BZ-9000 Keyence microscope. Confocal imaging was performed using a confocal laser scanning microscope (LSM880, Zeiss) equipped with a Plan Apochromat 10× / 0.45NA air or 63× / 1.4NA oil immersion objective. A multi-line argon laser was used for 488 nm excitation of AlexaFluor 488, and a HeNe laser was used for 561 nm excitation of AlexaFluor 594.

[0471] Immunostaining of human tissues

[0472] Formalin-fixed, paraffin-embedded tissues were sectioned at 6 μm and mounted on organosilane-coated slides (SIGMA, St. Louis, MO). Representative sections of the specimens were treated with hematoxylin / eosin and processed for immunocytochemistry. Immunocytochemistry for C1q (1:200, rabbit polyclonal; DAKO, Denmark) was performed on paraffin-embedded tissues as described previously. Sections were incubated for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. Single-label immunocytochemistry was performed using the Powervision method with 3,3-diaminobenzidine as the chromogen. Sections were counterstained with hematoxylin. An extensive neuropathology protocol (based on the recommendations of the Brain-Net Europe consortium; Acta Neuropathologica 115(5):497–507·2008) was used, including markers such as pTau (AT8), β-amyloid, pTDP-43, and α-synuclein.

[0473] Slice preparation for electrophysiology

[0474] We euthanized mice with 4% isoflurane, perfused with ice-cold sucrose cutting solution containing 234 mM sucrose, 11 mM glucose, 10 mM MgSO 4 , 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 0.5 mM CaCl 2 , and 26 mM NaHCO 3 , equilibrated with 95% O 2 and 5% CO 2 (pH 7.4), and decapitated. We prepared 250-μm-thick horizontal slices for thalamic recordings and coronal slices for neocortical recordings using a Leica VT1200 microtome (Leica Microsystems). Slices were incubated at 32°C for one hour and then incubated at 24-26°C in artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 10 mM glucose, 2.5 mM KCl, 2 mM CaCl2, 1.25 mM NaH2PO4, 1 mM MgSO4, and 26 mM NaHCO3, equilibrated with 95% O2 and 5% CO2 (pH 7.4). Thalamic slice preparation was performed as described.

[0475] Patch-clamp electrophysiology

[0476] Recordings were performed as described above. We used an Olympus microscope and an infrared camera to visually identify S1, nRT, and VB neurons using differential phase contrast optics. Recording electrodes made of borosilicate glass had a resistance of 2.5-4 MΩ when filled with intracellular solution. Access resistance was monitored in all recordings, and cells were included for analysis only when access resistance was <25 MΩ. In the presence of picrotoxin (50 μM, Sigma) and an internal solution containing 120 mM potassium gluconate, 11 mM EGTA, 11 mM KCl, 10 mM HEPES, 1 mM CaCl2, and 1 mM MgCl2 (pH adjusted to 7.4 with KOH (290 mOsm)), intrinsic and burst characteristics and spontaneous excitatory postsynaptic currents (EPSCs) were recorded. The potential was corrected to -15 mV liquid junction potential.

[0477] Spontaneous inhibitory postsynaptic currents (IPSCs) were recorded in the presence of kynurenic acid (2 mM, Sigma), and the internal solution contained 135 mM CsCl, 10 mM EGTA, 10 mM 685HEPES, 5 mM Qx-314 (N-ethyllidocaine bromide), and 2 mM MgCl2, with the pH adjusted to 7.3 with CsOH (290 mOsm).

[0478] Single-nucleus RNA-seq

[0479] Tissue dissection

[0480] We euthanized mice with 4% isoflurane, perfused them with ice-cold 1X PBS, and decapitated them. We prepared 300-μm-thick coronal sections using a Leica VT1200 microtome (Leica Microsystems) and placed them under a Zeiss SteREODiscovery.V8 stereomicroscope (Zeiss) for visually guided microdissection of the nRT and the adjacent relay thalamic nucleus (e.g., Figure 3A shown).

[0481] Two replicates of sham and mTBI groups were collected for this study. Replicate 1 contained tissue from n=5 sham-operated mice and n=6 mTBI mice. Replicate 2 contained tissue from n=4 sham-operated mice and n=4 mTBI mice.

[0482] Single nucleus isolation

[0483] Nuclei were isolated from the nRT / thalamus and cortex as previously described (62 and dx.doi.org / 10.17504 / protocols.io.6t8herw). Briefly, the tissue was placed in a pre-chilled Dounce tissue grinder with 1 mL of homogenization buffer containing 200 units of RNasin Plus ribonuclease inhibitor (Promega). Tissue samples were homogenized using a loose "A" pestle with 10 strokes and a tight "B" pestle with 15 strokes. The lysate was passed through a 40 μm FlowMi filter and the nuclei were precipitated at 500 RCF at 4°C. Fractions of the resulting supernatant containing cytoplasmic RNA were frozen for downstream analysis. The precipitated nuclei were resuspended in homogenization buffer, purified using an iodixanol gradient, and immediately used for snRNA-seq. Excess nuclei were cryopreserved in BamBanker (Wako Chemicals).

[0484] Single-nucleus RNA library construction and sequencing

[0485] SnRNA-seq libraries were processed using the Chromium Next GEM Single Cell 3'v3 Library Kit with Dual Indexing (10x Genomics) according to the manufacturer's instructions. For each sample, nuclei were diluted to 1,000 nuclei / μl in nuclei dilution buffer and 9,900 nuclei were loaded onto chromium, with targeted recovery of 6,000 nuclei. Replicate 1 and 2 nuclei were processed in different chromium rounds. Libraries were pooled based on the molar concentration of the libraries and sequenced using the S1 flow cell and v1 300 cycle kit on the Illumina NovaSeq 6000 system, with 28 cycles for read 1, 90 cycles for read 2, 10 cycles for index i7, and 10 cycles for index i5. Cell Ranger (4.0.0) (10X Genomics) was used to perform sample demultiplexing, barcoding, and single-cell gene UMI counting. Reads were mapped to mm10 (GENCODE vM23 / Ensembl 98, from 10x). From replicate 1, we recovered 2,337 nuclei from sham-operated mice with an average of 92,533 reads per cell, and 650 nuclei from mice with mTBI with an average of 162,363 reads per cell. From replicate 2, we recovered 3,891 nuclei from sham-operated mice with an average of 47,592 reads per cell, and 4,575 nuclei from mice with TBI with an average of 41,658 reads per cell, with a median of 2,200 genes per cell. Raw data are deposited in GEO under accession number [ 1 ].

[0486] Analysis of nuclear clustering

[0487] After processing with CellRanger, the data matrix was analyzed using Seurat. Prior to analysis, environmental RNA was removed using SoupX with default parameters. Potential doublets were removed from each GEM reaction using DoubletFinder. In replicate 1, 90 doublets were removed from sham surgery and 147 doublets were removed from TBI. In replicate 2, 28 doublets were removed from sham surgery and 181 doublets were removed from TBI. Nuclear doublets from DoubletFinder were used for downstream analyses. In addition to removing doublets, we also removed nuclei with greater than 1% expression of mitochondrial RNA. Expression was log-normalized, and the top 2000 features were used for PCA and downstream clustering and UMAP. Harmony was used to merge individual replicates. After Harmony correction, we observed no differences between replicates. Clusters were called using FindClusters and manually annotated using key lineage markers. GABAergic nuclei were again subclustered after selecting Slc17a7 / Slc17a6-negative neurons. Differentially expressed genes (DEGs) between clusters and between sham and mTBI were analyzed using the FinderMarkers function. P values ​​were calculated using the Wilcoxon rank-sum test. For expression visualization in the UMAP project, RNA expression values ​​were interpolated using the Markov Affinity-based Graph Imputation of cells (MAGIC).

[0488] Quantitative real-time PCR of cytoplasmic RNA

[0489] As previously described, a large amount of cytoplasmic RNA was extracted from each replicate sample (62). Briefly, 150 μL of the homogenate was mixed with 1.5 mL of Trizma (Zymo). The aqueous layer was retained, mixed with ethanol, and loaded onto a Zymo GC column (Zymo Quick RNA Mini Kit) according to the manufacturer's instructions for Zymo. RNA concentration was quantified using a spectrophotometer. 200 ng of each sample was used as input for cDNA synthesis using a SuperScript III First-Strand Synthesis kit (Invitrogen) using random hexamer primers according to the manufacturer's instructions. Specific target cDNAs were quantified using SSO Advanced Universal Sybr Green Supermix (BioRad) according to the manufacturer's instructions. Actin was used as an internal reference and relative expression was calculated using the δ-δCT method. Samples not treated with reverse transcriptase were used to determine background.C1qa-F 5′-ATGGAGACCTCTCAGGGATG-3′(SEQID NO:69), C1qa-R 5′-ATACCAGTCCGGATGCCAGC-3′(SEQ ID NO:70); NO:71); NO:74);C1ra-F:5′-GCCATGCCCAGGTGCAAGATCAA-3′(SEQ ID NO:75);C1ra-R:5′-TGGCTGGCTGCCCTCTGATG-3′(SEQ ID NO:76); NO:77);C1s-R:5′-GGTGGGTACTCCACAGG CTGGAA-3′(SEQ ID NO:78);C2-F:5′-CTCATCCGCGTTTACTCCAT-3′(SEQID NO:79). NO:80);C3-F:5′-AGCAGGTCATCAAGTCAGGC-3′(SEQ ID NO:81);C3-R:5′-GATGTAGCTGGTGTTGGGCT,-3′(SEQ IDNO:82);C4-F:5′-ACCCCCTAAATAACCTGG-3′(SEQ ID NO:83);C4-R:5′-CCTCATGTATCCTTTTTGGA-3′(SEQ ID NO:84);Hc-F:5′-AGGGTACTTTGCCTGCTGAA-3(SEQ IDNO:85);Hc-R:5′-TGTGAAGGTGCTCTTGGATG-3′(SEQ ID NO:86).

[0490] In the case of the ECoG (Environmental Protection Agency) and MUA (Municipal Affairs)

[0491] Equipment for simultaneous ECoG, MUA recording, and optical manipulation in freely behaving mice was custom-made in the Paz laboratory as described in [ 15 ]. In general, recordings were optimized for assessing somatosensory subnetworks (primary somatosensory cortex (S1), somatosensory ventrobasal thalamus (VB), and somatosensory reticular thalamic nucleus (nRT)). Cortical screws were implanted bilaterally in S1 (contralateral to the lesion: -0.5 mm posterior to bregma, -3.25 mm lateral; ipsilateral: +1.0–1.4 mm anterior to bregma, +2.5–3.0 mm lateral), centrally over the PFC (+0.5 mm anterior to bregma, 0 mm lateral), and in the right hemisphere over V1 (-2.9 mm posterior to bregma, +2.7 mm lateral). For MUA recordings in the VB, we implanted the electrode at −1.65 mm posterior to bregma and +1.75 mm lateral to the anterior cerebral cortex, with the fiber tip at 3.0 mm and the electrodes at 3.25 mm and 3.5 mm ventral to the cortical surface. For MUA recordings in the nRT, we implanted the electrode at −1.4 mm posterior to bregma and +2.1 mm lateral to the anterior cerebral cortex, with the fiber tip at 2.7 mm and the electrodes at 2.9 mm and 3.0 mm ventral to the cortical surface, respectively.

[0492] In vivo electrophysiology and behavior

[0493] Non-chronic MUA electrophysiological recordings were performed in freely behaving mice as described using a custom optode apparatus. ECoG and thalamic LFP / MU (local field potential / multiunit) signals were recorded using an RZ5 (TDT) and sampled at 1221 Hz, with thalamic MUA signals sampled at 24 kHz. Animals were continuously monitored using a camera synchronized with signal acquisition. We briefly anesthetized the animals with 2% isoflurane at the beginning of each recording to connect for recording. Each recording trial lasted 15-60 minutes. To control for circadian rhythms, we housed the animals with a regular light / dark cycle and recorded between approximately 9:00 am and 6:00 pm. All recordings were performed in the awake state. We verified the position of the optodes by histology after euthanasia of the mice, which did not experience sudden death and for which we were able to recover and process their brains.

[0494] Surgical implantation of chronic ECoG recording devices

[0495] The wireless telemetry equipment we used for chronic ECoG recordings was purchased from Data Sciences International (DSI). Following controlled cortical impact surgery, we implanted cortical screws bilaterally above S1 as described above. The battery / transmitter device was placed under the skin above the right shoulder. We began recording mice once they had recovered from surgery. Mice were individually housed in their home cages on a receiver that sent signals to an acquisition computer. ECoG signals were continuously recorded from up to eight mice simultaneously using Ponemah software (DSI) and sampled at 500 Hz.

[0496] Statistical analysis

[0497] Unless otherwise stated, all values ​​are given as mean values, and error bars are standard errors of the mean (SEM). Parametric and nonparametric tests were selected where appropriate and reported in the figure legends. Data were analyzed using MATLAB (SCR_001622), GraphPad Prism 7 / 8 (SCR_002798), ImageJ (SCR_003070), Ponemah / NeuroScore (SCR_017107), pClamp (SCR_011323), and Spike2 (SCR_000903).

[0498] Image analysis and cell quantification

[0499] We selected regions of interest (ROIs) for S1, nRT, and VB from 10x Keyence microscope images opened in ImageJ (SCR_003070). To ensure that each ROI covered the same area on the ipsilateral and contralateral sides of the injury site, the first ROI was copied and repositioned on the contralateral hemisphere. The images were then converted to 8 bits. The upper threshold was adjusted to a maximum value of 255, and the lower threshold was increased from 0 until the pixel appearance most closely matched the fluorescent staining of the original image. Particle analysis was performed on the ROI using the same threshold boundaries for all sections with the same staining. The integrated density ratio for each brain region was calculated by dividing the ipsilateral integrated pixel density by the contralateral integrated pixel density. The integrated density ratios of the three sections for each animal were averaged to obtain a single average ratio for each brain region for each animal.

[0500] NeuN-stained sections were subjected to nRT cell counting. nRT was outlined in ImageJ (SCR_003070), and we performed manual cell counting of neuronal somata using the manual counter plugin.

[0501] Analysis of electrophysiological characteristics

[0502] Input resistance (Rin) and membrane time constant (τm) were measured by membrane hyperpolarization in response to low-intensity current steps (-20 to -60 pA). The reported mean and SEM of the base strength were calculated based on the current that first generated at least one action potential during each recording stimulation. All data were analyzed using GraphPad 755 Prism 7 (SCR_002798) with a Mann-Whitney test at α = 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0503] Cumulative probability distributions were generated from 11 sham-operated nRT neurons and 9 TBI neurons in MATLAB (SCR_001622) using 200 events randomly selected from each cell.

[0504] Detection of spindle waves and epileptic spike events in ECoG

[0505] We used the MorletWavelet function to detect spindle waves in the frequency range of 8–15 Hz. We applied a threshold of [1.5 times the SD + 1 times the mean] of the ECoG power and detected all events above this threshold that lasted for at least 0.5 seconds (Figure 16, Figure 18 ). All detected events were visually verified by a scientist blinded to the groups. The onset and offset times of the spindle wave events were extended to the nearest cycle of the 0 crossing before and after the threshold. The spindle wave amplitude was calculated from the average amplitude of the spindle wave (8-15 Hz) power between the onset and offset times, divided by the RMS of the ECoG signal, and averaged for each mouse. The frequency of the spindle wave was determined by extracting the peak frequency from the FFT amplitude of each spindle wave event and then calculating the average internal frequency for each mouse. False positive events containing epileptic spikes (defined as events exceeding the threshold 7 times SD + 1 times the baseline mean, Figures 16 and 17) were rejected after visual verification by a scientist blinded to the groups.

[0506] Sleep scoring and data analysis were performed using Spike2 (version 7.20, Cambridge Electronic Design, Cambridge, UK) and Python 3.7 (Python Software Foundation). 5-second periods were automatically scored (Spike2) and designated as wakefulness, REM sleep (REM), and NREM / slow-wave sleep. Automatic scoring was further visually inspected by an experienced scientist who was unaware of the treatment groups. If the ratio of ECoG delta (δ, 1.5-4 Hz) to total power (1.5-80 Hz) was above the threshold for no motor activity, the period was designated as NREM sleep. On day 20 or 21 after mTBI / sham surgery, sleep spindle analysis was performed for a 12-hour (7am–7pm) period during NREM sleep. Epileptic spikes were analyzed within the same time period. Recordings during movement were not analyzed because it was difficult to reliably distinguish between motion artifacts and epileptic spikes (Figure 17).

[0507] Anti-C1q antibodies

[0508] The anti-C1q antibody M1 described herein demonstrates robust binding to mouse C1q and can inhibit functional complement activity in the serum of multiple animal species (Lansita et al., 2017). Previous studies have reported a lack of toxicity in rodents and monkeys (Lansita et al., 2017) and demonstrated in vivo inhibition in several mouse models (McGonigal et al., 2013, Vukojicic et al., 2019). Twenty-four hours after TBI or sham surgery, mice were intraperitoneally administered an injection of anti-C1q antibody M1 or a mouse IgG1 isotype control antibody and continued to receive treatment every three days (4 days after TBI, 7 days after TBI, etc.) for three weeks.

[0509] Treatment modalities and tissue lysis

[0510] For PK studies, mice received sham or TBI surgery on day 0 and were treated intraperitoneally with 100 mg / kg anti-C1q antibody (M1) or isotype control on days 1 and 4. Mice were perfused with PBS on day 5. Plasma and brain (ipsilateral and contralateral) were collected and snap-frozen. The cells were homogenized in a Qiagen TissueLyser with 7 mm steel beads at 30 Hz for two minutes in a 1:10 w / v BupH 5 buffer solution. TM The brain (excluding the olfactory bulb and cerebellum) was lysed in Tris-buffered saline (Thermo Scientific 28379) + protease inhibitor cocktail (Thermo Scientific A32963). The lysate was then centrifuged at 17,000 x g for 20 minutes. The supernatant was used for ELISA assay.

[0511] Pharmacokinetic (PK) and pharmacodynamic (PD) ELISA assays

[0512] Levels of free anti-C1q drug M1 (PK), free C1q, total C1q, C1s, and albumin were measured using a sandwich ELISA. Black 96-well plates (Nunc 437111) were coated overnight at 4°C in bicarbonate buffer (pH 9.4) with 75 μL of the respective capture protein / antibody: human C1q protein (Complement Tech) for PK, mouse monoclonal anti-C1q (Abcam, ab71940) for free C1q, rabbit polyclonal anti-C1q (Dako, A0136) for C1s, and rabbit polyclonal anti-mouse C1s (LSBio, C483829). The following day, the plates were washed with dPBS (Dulbecco's Phosphate-Buffered Saline, pH 7.4) and blocked with dPBS containing 3% bovine serum albumin (BSA). Standard curves were prepared using purified proteins in assay buffer (dPBS containing 0.3% BSA and 0.1% Tween 20). Prepare samples with appropriate dilutions in assay buffer. Remove blocking buffer from the plate by tapping. Add standards and samples in duplicate with 75 μL per well and incubate at room temperature with 300 rpm shaking for one hour for PK measurement. For complement determination, samples were incubated overnight at 4 ° C, then incubated at 37 ° C for 30 minutes, and then incubated at room temperature for 1 hour. The plate was then washed three times with dPBS containing 0.05% Tween20, and 75 μL alkaline phosphatase-conjugated secondary antibodies (goat anti-mouse IgG for PK, M1 for free C1q, rabbit polyclonal anti-C1q for total C1q, rabbit polyclonal anti-C1s for C1s) were added to all wells. The plate was incubated at room temperature with shaking for one hour, washed three times with dPBS containing 0.05% Tween20, and developed using 75 μL alkaline phosphatase substrate (Life Technologies, T2214). After 20 minutes at room temperature, the plate was read using a photometer. Albumin assays were performed using a matched pair of antibodies from Abcam (ab210890), followed by detection using an avidin-AP secondary antibody. Standards were fitted and unknown concentrations determined using a 4PL logistic fit. Analyte levels were corrected for the dilution factor.

[0513] Example 2: Secondary C1q expression is associated with chronic inflammation, neurodegeneration, and synaptic dysfunction in the thalamus To

[0514] To determine the secondary long-term effects of mTBI, we induced a mild cortical impact injury (MSI) to the right pr...

Claims

1. Use of an anti-C1q antibody for the manufacture of a medicament for preventing, reducing the risk of, or treating epilepsy induced by traumatic brain injury (TBI), wherein the anti-C1q antibody comprises: a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO:5, HVR-L2 having the amino acids of SEQ ID NO:6, and HVR-L3 having the amino acids of SEQ ID NO:7; and A heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO:9, HVR-H2 having the amino acid sequence of SEQ ID NO:10, and HVR-H3 having the amino acid sequence of SEQ ID NO:

11.

2. The use according to claim 1, wherein the TBI-induced epilepsy is post-traumatic epilepsy.

3. The use according to claim 1 or 2, wherein the medicament is for administration during an attack or within the first 4 weeks after an attack.

4. The use according to claim 1 or 2, wherein the medicament is for administration during an attack or within the first week after an attack.

5. The use according to claim 1 or 2, wherein the drug is for administration during an attack or within 24 hours after an attack.

6. The use of claim 1 or 2, wherein the medicament is for administration during an attack or within 1, 2, 3, 4, 5 or 6 hours after an attack.

7. The use according to claim 1 or 2, wherein the anti-C1q antibody inhibits synaptic loss induced by seizures.

8. The use according to claim 1 or 2, wherein the medicament is used for administration to a subject suffering from anoxic brain injury, brain infection, stroke or genetic syndrome.

9. The use according to claim 8, wherein the brain infection is encephalitis, meningitis, mesial temporal sclerosis or brain tumor.

10. The use according to claim 8, wherein the anti-C1q antibody inhibits synaptic loss induced by traumatic brain injury, anoxic brain injury, brain infection, stroke or genetic syndrome.

11. The use of claim 8, wherein the medicament is for administration during or within the first 4 weeks after traumatic brain injury, anoxic brain injury, brain infection or stroke.

12. The use according to claim 8, wherein the medicament is for administration during or within the first week after traumatic brain injury, anoxic brain injury, brain infection or stroke.

13. The use according to claim 8, wherein the medicament is for administration within 24 hours during or after traumatic brain injury, anoxic brain injury, brain infection or stroke.

14. The use of claim 8, wherein the medicament is for administration within 1, 2, 3, 4, 5 or 6 hours during or after traumatic brain injury, anoxic brain injury, brain infection or stroke. 15 . The use according to claim 1 or 2 , wherein the anti-C1q antibody inhibits the interaction between C1q and autoantibodies, between C1q and C1r, or between C1q and C1s.

16. The use according to claim 1 or 2, wherein the anti-C1q antibody promotes the clearance of C1q from circulation or tissues.

17. The use according to claim 1 or 2, wherein the anti-C1q antibody has a dissociation constant (K) ranging from 100 nM to 0.005 nM or less than 0.005 nM. D ).

18. The use of claim 1 or 2, wherein the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 20:1 to 1.0:1 or less than 1.0:

1.

19. The use of claim 18, wherein the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 6:1 to 1.0:1 or less than 1.0:

1.

20. The use of claim 19, wherein the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 2.5:1 to 1.0:1 or less than 1.0:

1.

21. The use according to claim 1 or 2, wherein the anti-C1q antibody specifically binds to C1q and neutralizes the biological activity of C1q.

22. The use according to claim 21, wherein the biological activity is (1) binding of C1q to autoantibodies, (2) binding of C1q to C1r, (3) binding of C1q to C1s, (4) binding of C1q to IgM, (5) binding of C1q to IgG, (6) binding of C1q to phosphatidylserine, (7) binding of C1q to pentraxin-3, (8) binding of C1q to C-reactive protein (CRP), (9) binding of C1q to globular C1q receptor (gC1qR), (10) binding of C1q to complement receptor 1 (CR1), (11) binding of C1q to β-amyloid protein, (12) binding of C1q to calreticulin, (13) binding of C1q to apoptotic cells, or (14) binding of C1q to B cells.

23. The use of claim 21, 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) microglia activation, (10) immune complex formation, (11) phagocytosis of synapses or nerve endings, (12) activation of complement receptor 3 (CR3 / C3) expressing cells or (13) neuroinflammation.

24. The use according to claim 23, wherein the CH50 hemolysis comprises CH50 hemolysis of humans, mice, rats, dogs, rhesus monkeys and / or cynomolgus monkeys.

25. The use according to claim 23, wherein the anti-C1q antibody is capable of neutralizing 50% to 90% of CH50 hemolysis.

26. The use of claim 23, wherein the anti-C1q antibody is capable of neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml or less than 20 ng / ml.

27. The use according to claim 1 or 2, wherein the anti-C1q antibody is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antigen-binding fragment thereof.

28. The use according to claim 27, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment or a single-chain antibody molecule.

29. The use according to claim 1 or 2, wherein the anti-C1q antibody is coupled to a labeling group.

30. The use according to claim 29, wherein the labeling group is an optical label, a radioisotope, a radionuclide, an enzymatic group, a biotinyl group, a nucleic acid or an enzyme.

31. The use according to claim 30, wherein the optical label is a fluorescent label.

32. The use according to claim 30, wherein the nucleic acid is an oligonucleotide.

33. The use of claim 1 or 2, wherein the anti-C1q antibody comprises a light chain variable domain comprising an amino acid sequence having about 95% homology to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, and wherein the light chain variable domain comprises HVR-L1 having an amino acid sequence of SEQ ID NO: 5, HVR-L2 having amino acids of SEQ ID NO: 6, and HVR-L3 having amino acids of SEQ ID NO:

7.

34. The use of claim 33, wherein the light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 4 and 35-38.

35. The use of claim 1 or 2, wherein the anti-C1q antibody comprises a heavy chain variable domain comprising an amino acid sequence having about 95% homology to an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, and wherein the heavy chain variable domain comprises HVR-H1 having an amino acid sequence of SEQ ID NO: 9, HVR-H2 having amino acids of SEQ ID NO: 10, and HVR-H3 having amino acids of SEQ ID NO:

11.

36. The use according to claim 35, wherein the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 8 and 31-34.

37. The use according to claim 27, wherein the antigen-binding fragment comprises a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40.

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