TITLE OF THE INVENTION

COMPOSITIONS AND METHODS FOR TREATING CENTRAL NERVOUS

SYSTEM INJURY

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under

1P20GM109040-01 awarded by the National Institute of Health. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/409, 184 filed October 17, 2016, the contents of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Currently, ischemic stroke is the fifth leading cause of death in the U.S. and is also a major cause of long-term disability in the U.S and worldwide. Traumatic brain injury and spinal cord injury are also major causes of disability worldwide, especially among children and young adults, and such injuries are also prominent types of combat-related injury.

Following onset of cerebral ischemia, many stroke patients show reperfusion of their infarct either spontaneously or as a secondary effect of thrombolytic therapy. Cerebral reperfusion initiates a cascade of pathophysiological events that cause secondary injury, which can lead to greater tissue damage and more severe functional and cognitive deficit. Clinical observation and experimental studies indicate a central role for complement in the propagation of ischemia reperfusion injury (IRI) in both central nervous system (CNS) and in non-CNS tissue.

Despite efforts to develop effective strategies for treatment of stroke, the field remains hampered by the necessity to initiate treatment immediately after onset. Thus there is a need in the art for improved compositions and methods for treating stroke. The present invention satisfies this unmet need.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition for treating central nervous system injury comprising: (a) a targeted inhibitor molecule comprising a targeting portion and an inhibitor portion, wherein the molecule inhibits complement signaling; and (b) a thrombolytic agent.

In one embodiment, the composition provides a targeted inhibitor molecule wherein the targeting portion comprises a CR2 protein or fragment thereof.

In one embodiment, the inhibitor portion comprises at least one, or a fragment thereof, selected from the group consisting of FH, DAF, MCP, CD59, Crry, MAp44, and CR1.

In one embodiment, the composition comprises a thrombolytic agent wherein the thrombolytic agent is t-PA.

In one aspect, the present invention provides a method for treating central nervous system injury in a subject comprising: (a) administering to the subject a therapeutically effective amount of a composition comprising a targeted inhibitor molecule comprising a targeting portion and an inhibitor portion, wherein the molecule inhibits complement signaling; and (b) providing rehabilitation therapy to the subject.

In one embodiment, the injury is ischemic stroke. In one embodiment, the injury is traumatic brain injury. In one embodiment, the injury is spinal cord injury.

In one embodiment, the targeting portion comprises CR2 or fragment thereof. In one embodiment, the inhibitor portion comprises at least one selected from the group consisting of FH, DAF, MCP, CD59, Crry, MAp44, and CR1.

In one embodiment, the rehabilitation therapy comprises at least one therapy selected from the group consisting of cognitive and motor therapy.

In one aspect, the present invention provides a method for treating central nervous system injury in a subject comprising: (a) administering to the subject a therapeutically effective amount of a composition comprising a targeted inhibitor molecule comprising a targeting portion and an inhibitor portion, wherein the molecule inhibits complement signaling; and (b) administering to the subject a composition comprising a thrombolytic agent.

In one embodiment, the injury is ischemic stroke. In one embodiment, the injury is traumatic brain injury. In one embodiment, the injury is spinal cord injury.

In one embodiment, the targeting portion comprises CR2 or fragment thereof. In one embodiment, the targeted inhibitor molecule comprises at least one selected from the group consisting of FH, DAF, MCP, CD59, Crry, MAp44, and CR1.

In one embodiment, thrombolytic agent is t-PA.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1, comprising Figure 1 A through Figure ID is a set of images depicting CR2fH treatment after Middle Cerebral Artery Occlusion (MCAO). Figure 1 A is an image depicting the percentage of animals dying due to hemorrhage (of total acute deaths) compared to controls. Population proportion statistic. *p<0.01. Figure IB is an image depicting CR2fH treatment significantly reducing hemoglobin content in the ipsilateral hemisphere 12 hours after MCAO compared to vehicle. Student' s t-test, n=4/group **p<0.01. Figure 1C is an image depicting the representative images of Nissl stained brain sections showing large hemorrhagic transformation and intracerebral hemorrhage in vehicle controls but not CR2fH treated animals (90 minutes after administration). Figure ID is an image depicting the combination of CR2fH and rehabilitation significantly enhanced survival at 15 days after MCAO. Survival curve showing that CR2fH treatment 90 min after reperfusion alone or in combination with rehab significantly reduced post-stroke mortality up to 15 days after injury. Combination of rehab and CR2fH administered 6 or 24 hours after MCAO also significantly improved survival compared to vehicle only. Kaplan-Meyer Curve. N= 7-21/group. (*)p<0.05 compared to vehicle. (PBS, Phosphate buffered saline; R, rehab; R, No rehab; fB+/-, factor B.

Figure 2, comprising Figure 2A through Figure 2D, is a set of images depicting the results of experiments from CR2fH treatment (Enriched Environment + skilled handling). Figure 2A depicts a set of images representative of T2-weighted MRI images at days 4 and 14 after MCAO showing large hyper-intense lesions in vehicle and rehab animals compared to CR2fH treated animals with or without rehab. Figure 2B is an image illustrating the quantification of (Figure 2A) showing significant reduction of hyper-intense lesion volume with CR2fH treatment and not rehab. ANOVA + Tukey (multiple comparisons. n=7-8/group. *p<0.05, **p<0.01, ***p<0.001. Figure 2C is a set of images illustrating Nissl stained brains showing secondary scarring in the ipsilateral hemisphere 15 days after MCAO that was inhibited by acute CR2fH therapy, (cl): Nissl stained brain slices, (c2) 3D-rendering of lesion (in red) on a brain showing the location of cortex (yellow), basal ganglia (green), white matter (grey), and hippocampus (blue). Mapping of lesion at different stereotactic coordinates (relative to Bregma) to the Paxinos brain atlas showing the frequency of scarring over serial brain sections for animals within each group (c3). Heat maps map the frequency of overlapping lesions from 8 animals per group. Figure 2D depicts an image illustrating the quantification of lesion volume confirming histologically that CR2fH reduces secondary scarring 15 days after MCAO with minimal contribution of rehab alone to reduction of scarring. ANOVA + Tukey. n=8/group. **p<0.01, ***p<0.001. (EE: enriched environment)

Figure 3, comprising Figure 3 A through Figure 3M, is a set of images depicting the combination of CR2fH treatment and rehabilitation. Figure 3B is a set of images depicting CR2fH administered 90 minutes after ischemia with or without rehab. Two-way ANOVA, Bonferroni. n=12-16/group (n=21 for vehicle group). *p<0.05

***p<0.001. Figure 3C through Figure 3D is a set of images depicting CR2fH improved recovery of functional deficits when administered 6 hours (Figure 3C) or 24 hours (Figure 3D) after MCAO. Figure 3E is an image illustrating the comparison of neurological deficits at day 15 after injury showing that CR2fH significantly improved chronic functional recovery compared to vehicle. Comparisons made against vehicle group. Kruskal-Wallis test with Dunn's multiple comparisons. n=12-16/group (n=21 for vehicle group), ns: not significant. *p<0.05. ***p<0.001. ****p<0.0001. Figure 3F is an image depicting the pair-wise comparison of functional recovery between days 2 and 15 across the different groups. Two-way ANOVA with Bonferroni. n=12-16/group (n=21 for vehicle group). *p<0.05. Figure 3G is a set of images depicting animals treated with rehab, and animals co-treated with CR2fH after MCAO. Two-way ANOVA with

Bonferroni. n=16 (vehicle), 12 (CR2fH 90 minutes), 7 (CR2fH 6 hours and 24 hours). *p<0.05.***p<0.001. Figure 3H and Figure 31 is a set of images that illustrate the first week of recovery, CR2fH +/- rehab significantly reduced forelimb asymmetry on corner test compared to vehicle (Figure 3H). Figure 31 is an image depicting open field locomotor activity showing a similar pattern as in (Figure 3H). No difference was seen in the percent time spent at center controlling for potential difference in anxiety levels between the groups (Figure 3J). Two-way ANOVA with Bonferroni. n=12-16/group (n=21 for vehicle group). *p<0.05. **p<0.01. ***p<0.001. Figure 3K through Figure 3M depicts a set of images using pasta handling task to assess forelimb skilled movement compared to vehicle controls. Two-way ANOVA with Bonferroni. n=8/group. *p<0.05.

Figure 4, comprising Figure 4 A through Figure 4F is a set of images depicting improvement in cognitive performance after MCAO with rehab or CR2fH. Figure 4A through Figure 4C is a set of images depicting CR2fH alone or in combination with rehab significantly improving spatial learning (days 9-11 after MCAO) and retention of learned memory (day 15 post-MCAO) compared to vehicle controls as assessed by the total path length before reaching the target hole (Figure 4A) or the number of error pokes (Figure 4C). Co-treatment with CR2fH and rehab also reduced path length and number of errors significantly compared to rehab alone during learning. Two-way ANOVA, Bonferroni. N=8/group. *p<0.05. ***p<0.001. Figure 4D is an image that depicts passive avoidance task of animals treated with CR2fH+/- rehab have better memory retention (longer time to enter the shock chamber) compared to vehicle and rehab alone starting 7 days after MCAO. Two-way ANOVA, Bonferroni. N=8/group. *p<0.05. **p<0.01. ***p<0.001. Figure 4E is an image depicting the principle component analysis of the performance on the different motor and cognitive tasks displayed in Figure 2 and Figure 3, showing that 3 principle components (PC1-PC3) can explain 99.2% of the variance. Individual animal data including shams were plotted against the 3 PCs showing that CR2fH combined with rehab was most efficient at bringing the animals closer to sham compared to either single therapy. Figure 4F is an image depicting the result of PC A analysis performed only on motor (right) or cognitive tasks (left) indicate that the effect of rehab was more pronounced on cognitive rather than motor tasks. Two principal components explaining around 90% of the variance were plotted.

Figure 5, comprising Figure 5A through Figure 5G is a set of images depicting CR2fH treatment blocks an inflammatory response after MCAO. Figure 5A and Figure 5B depicts a set of images illustrating sustained neuroinflammatory response manifested by C3d and IgM deposition 15 days after MCAO was inhibited by single acute administration of CR2fH (90 minutes after MCAO). Shown in Figure 5 A are the lesion locations for each column, and in (Figure 5B) the different treatment groups. Green: IgM. Red: C3d. Blue: DAPI. Figure 5C and Figure 5D is a set of images illustrating the quantification of (Figure 5B) for IgM (Figure 5C) and C3d (Figure 5D) deposition showing that CR2fH significantly reduce deposition of both markers on the inflamed ipsilateral endothelium. Rehab alone resulted in a mild though significant reduction in IgM and C3d deposition by 15 days after MCAO. ANOVA. N=5 animals (4 fields per animal chosen from the peri-infarct lesion). *p<0.05. ****p<0.0001. Figure 5E is a set of images illustrating GFAP immunofluorescence staining showing extensive astrogliosis in the peri-infarct area 15 days after MCAO that was significantly inhibited with CR2fH therapy and not rehab alone. Figure 5F is a set of images illustrating Mac2 immunofluorescence staining showing extensive gliosis and proliferation of Ml- polarized (inflammatory type microglia) in the peri-lesional area of vehicle and rehab animals that was significantly inhibited by CR2fH therapy. Figure 5G is a set of images depicting the quantification of (Figure 5E) and (Figure 5F) showing that despite CR2fH more robustly reduces astrocytosis and microgliosis, rehab does have a milder through significant effect on reducing inflammatory phenotype 15 days after stroke. ANOVA. N=5 animals (3 fields per animal). *p<0.05. **p<0.01. ***p<0.001.

Figure 6, comprises Figure 6 A through Figure 6M is a set of images depicting CR2fH removing regenerative mechanisms allowing maximal effect of rehab therapy. Figure 6A is a set of images depicting Dcx immunostaining of perilesional hippocampi (quantified in Figure 6B) showing significant increase in the number of neuroblasts migrating to the ipsilateral hippocampus 15 days after injury with rehab, CR2fH or combination therapy. ANOVA. N=6 animals (2 fields each). ***p<0.001. Figure 6C through Figure 6F is a set of images depicting the immunostaining for markers of regeneration and remodeling including dendritic arborization (MAP2), synaptic density (PSD-95) and axonal growth (GAP-43) of full brain slices. ANOVA. N=6 animals (full hemispheres quantified). *p<0.05. **p<0.01. ***p<0.001. Figure 6G is a set of images illustrating co-staining for GAP-43 and GFAP showing astrogliosis inhibits the regrowth of regenerating (GAP-43+) axons into the perilesional area. Figure 6H is a set of images depicting the quantification of GAP-43 density in the peri-lesional area showing that vehicle and rehab animals show a significant reduction in GAP43 density compared to CR2fH+/- rehab surrounding the areas of astrogliosis (x-axis = distance of the quantified cube relative to the center of the astrogliotic scar). Figure 61 is as set of images depicting representative 3D-rendered fields from (Figure 6H) at different positions relative to the astrogliotic scar. Figure 6J is an image depicting notable reduction in GAP43 near the areas of astrogliosis in vehicle-treated animals despite comparable neuronal density across the boundaries of the scar (Neurotrace, cyan). Figure 6K is a set of images depicting the representative western blots showing levels of neuronal growth factor (BDNF; Figure 6K1) and Tumor necrosis factor alpha (TNF-a; Figure 6K1) across the different groups normalized to house-keeping controls (beta-actin and GAPDH respectively). Figure 61 is set of images depicting both CR2fH and rehab alone resulted in significant increase in the levels of BDNF in the ipsilateral hemisphere compared to vehicle. ANOVA. n=6/group. *p<0.05. ***p<0.001. Figure 6M is an image illustrating rehab did not influence the levels of TNF-alpha whether alone or in combination with CR2fH treatment. ANOVA. n=5/group. **p<0.01. ***p<0.001.

Figure 7, comprises Figure 7A through Figure 7C is a set of images depicting immunofluorescence staining of post-mortem human brain sections for IgM (green) and C3d (red) from patients who died from acute stroke (24-72 hours after onset). Figure 7A is a set of images illustrating significant deposition of C3d and IgM

specifically in the ipsilateral hemisphere (ischemic penumbra) and not in the contralateral brain from the same patients. Figure 7B is a set of images illustrating the 3D-rendering of a high magnification field from patient FIB 1 showing co-localization of C3d and IgM 24 hours after stroke in the ischemic penumbra. (HB l : died 24 hours after stroke, HB2: 24- 48 hours, HB3 : 72 hours). Figure 7C is an image depicting the quantification of (Figure 7A) showing significantly higher IgM and C3d in the ischemic vs. control hemisphere. Pairwise Two-way ANOVA, Bonferroni. n=3. *p<0.05. **p<0.01.

Figure 8, comprises Figure 8A through Figure 8D is a set of images depicting CR2fH (90 minutes after MCAO) in female mice and aged male mice (18 months old). Figure 8A is an image illustrating CR2fH alone or in combination with rehab significantly reduced neurological deficits in aged mice compared to vehicle; CR2fH with rehab was significantly better compared to rehab only (p<0.05). Two-way ANOVA, Bonferroni. N=7/group. *p<0.05. **p<0.01. Figure 8B is an image illustrating survival was significantly improved by CR2fH +/- rehab in aged animals after MCAO. Experiments were terminated at day 10 due to loss of all vehicle controls. Log-rank (Mantel-Cox) test. N=7/group. *p<0.05. Figure 8C and Figure 8D is a set of images illustrating CR2fH improved neurological deficits and survival of adult female mice through 15 days after MCAO. Figure 8C is an image depicting two-way ANOVA,

Bonferroni. N=7/group. *p<0.05. **p<0.01. Figure 8D is an image depicting log-rank (Mantel-Cox) test. N=7/group. p=0.057.

Figure 9, comprising Figure 9A and Figure 9B depicts the neurological deficit scores (Figure 9A) and forelimb laterality (Figure 9B) showing animals treated with t-PA (30 minutes after high-dose emboli), CR2fH (30 minutes after high-dose emboli), and t-PA combined with CR2fH (30 minutes after high-dose emboli). Two-way ANOVA. N=6/group. *p<0.05. ***p<0.001.

Figure 10 is a graph showing the performance of mice on spatial learning task (Barnes maze) during chronic recovery from TBI. At 2 months after TBI, animals received 3 doses of vehicle or CR2Crry every other day and then assigned to

rehabilitation (enriched environment or regular housing). Animals treated with CR2Crry showed a significantly better learning curve (shorter path length during learning days 83- 87) and better retention of learned memory compared to vehicle or rehab alone at 4 weeks after treatment. Repeated measure two-way ANOVA. ***p<0.001. N=7/group. Combination of rehabilitation with CR2Crry also resulted in better cognitive recovery compared to rehabilitation alone. Repeated measure two-way ANOVA. **p<0.01.

N=7/group.

Figure 11, comprising Figure 11 A through Figure 11C depicts the design of CR2fH expression plasmid (Figure 11 A), the domains of the CR2fH protein with the signal peptide (Figure 1 IB), and the domains of the mature CR2fH protein (Figure 11C).

Figure 12 depicts the amino acid sequence of human CR2 (referred to as

SEQ NO: 1).

Figure 13 depicts exemplary amino acid sequence of mouse CR2 (SEQ ID

NO: 2).

Figure 14 depicts the amino acid sequence of an exemplary human CR2-

FH construct.

Figure 15 depicts an amino acid sequence of an exemplary mouse CR2-

FH construct.

Figure 16 depicts an exemplary nucleic acid sequence and amino acid sequence for mouse CR2-Crry.

DETAILED DESCRIPTION

The present invention provides compositions and methods for treating central nervous system injury, including, but not limited to, stroke, traumatic brain injury, and spinal cord injury. For example, the present invention is based upon the discovery that complement inhibition significantly reduces acute mechanisms of degeneration following stroke. For example, it is demonstrated herein that complement inhibition reduces infarct volume and improves functional recovery after stroke. Further, it is demonstrated herein that complement inhibition can be used as an adjuvant therapy in combination with standard stroke therapies to improve patient outcome. Therefore, the present invention provides methods for improving motor recovery, cognitive recovery, and survival after injury to the central nervous system.

In one aspect, the method provides for the use of a complement inhibitor to enhance the response and efficacy of rehabilitation therapy (both cognitive and motor) following central nervous system injury. It is demonstrated herein that a targeted complement inhibitor that inhibits the complement signaling significantly improves rehabilitation-induced motor and cognitive recovery as measured up to 15 days after stroke. The method provides for an effective treatment even when the complement inhibitor is administered as late as about 90 minutes, 2 hours, 4 hours, 6, hours, or 24 hours after injury, which is an improvement over the standard of care where t-PA must be administered with 3 hours of stroke. Thus, the present invention allows for an increase in the available treatment window for central nervous system injury.

In one aspect, the present invention provides for compositions and methods related to the use of targeted complement inhibition as an adjuvant therapy in combination with a thrombolytic agent to improve outcome after central nervous system injury.

A potential problem in the translation of a complement inhibitor strategy to the clinic is the immunosuppressive effect of systemic complement inhibition. Also, complement has important roles in homeostatic and physiological functions such immune complex catabolism, clearance of dead and dying cells, tissue repair, modulation of adaptive immunity, neuroregenerative processes and host defense. Other important concerns regarding the use of systemic complement inhibition relate to efficacy and biodistribution. An approach to alleviate the concerns of systemic inhibition described herein specifically targets complement inhibition to sites of complement activation. In this approach, a fragment of C receptor 2 (CR2) that recognizes the C3d activation product is linked to a complement inhibitor. It is demonstrated that site-specific targeting of a complement inhibitor obviates the need for systemic inhibition and increases bioavailability and efficacy, without affecting susceptibility to infection, unlike systemic complement inhibition. Definitions

Unless defined otherwise, 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 be used in the practice or testing of the present invention, the preferred methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%), ±5%), ±1%), or ±0.1%) from the specified value, as such variations are appropriate to perform the disclosed methods.

The term "subacute phase" is used herein to describe the period following the incident of a stroke that includes 7 days following an ischemic event or injury.

There term "in combination with" is used herein to indicate combining treatments either concurrently or at a timing which includes first administering a therapeutic agent and then administering rehabilitation or vice versa wherein the timing between initializing one and initializing the other is not limited.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

The terms "effective amount" and "pharmaceutically effective amount" or "therapeutically effective amount" refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of a sign, symptom, or cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The term "fusion protein" used herein refers to two or more peptides, polypeptides, or proteins operably linked to each other.

An "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. In some embodiments, the individual is human. In some embodiments, the individual is an individual other than human. The term "inhibit," as used herein, means to suppress or block an activity or function relative to a control value. Preferably, the activity is suppressed or blocked by 10% compared to a control value, more preferably by 50%, and even more preferably by 95%.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified

polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

The term "pharmaceutically acceptable" as used herein, refers to agents that, within the scope of sound medical judgment, are suitable for use in contact with tissues of human beings and/or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The terms "subject," "patient," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

The term "sub-therapeutic" as used herein means a treatment at a dose known to be less than what is known to induce a therapeutic effect.

The term "therapeutic" as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

The term "therapeutic agent" use herein refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if its administration to a relevant population is statistically correlated with a desired or beneficial therapeutic outcome in the population, whether or not a particular subject to whom the agent is administered experiences the desired or beneficial therapeutic outcome.

The term "therapeutically effective amount" as used herein, means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition (e.g., host versus graft disease). In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that particular subjects may, in fact, be "refractory" to a "therapeutically effective amount." To give but one example, a refractory subject may have a low bioavailability such that clinical efficacy is not obtainable. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective agent may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear

polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description

This invention describes a therapeutic composition and method related to the use of targeted complement inhibition as an adjuvant therapy in combination with one or more treatment regimens to improve outcome after central nervous system injury such as ischemic stroke, traumatic brain injury, or spinal cord injury. The present invention relates to compositions and methods for improving the recovery from ischemic stroke, and restoring cerebral function by reducing the risk of stroke-induced damage. In one embodiment, the method comprises administering to a subject a composition comprising a complement-targeted inhibitor in combination with

rehabilitation for use in enhancing recovery following central nervous system injury. In certain embodiments, the complement inhibitor is a composite molecule comprised of a targeting portion and an inhibitor portion wherein the composite molecule targets complement pathways. In certain embodiments, the complement-targeting portion comprises CR2.

The present invention relates to compositions and methods for improving the recovery from ischemic stroke, and restoring cerebral function reducing the risk to the recipient relating to the side effects around ischemic stroke including those involving complement pathways.

In one embodiment, the method comprises administering a composition comprising a complement-targeted inhibitor in combination with rehabilitation therapy. In some instances, the rehabilitation therapy is motor and cognitive therapy. In one embodiment, rehabilitation therapy comprises environmental enrichment.

In one aspect, the present invention relates to a composition used to treat a subject that has suffered an ischemic stroke, traumatic brain injury, or spinal cord injury. In one embodiment, the composition modulates signaling of complement pathways. In certain instances, the composition of the present invention comprises a composite molecule comprising a targeting portion and an inhibiting portion. In particular, the targeting portion directs the compound of the present invention to complement pathways and the inhibiting portion directs the compound to inhibit complement signaling. In some instances, the targeting portion is CR2. In some instances, the inhibiting portion is selected from a list comprising but not limited to Factor H (FH), Crry, DAF, MCP, MAp44, and CRl .

In one embodiment, the composition comprises a composite molecule and a thrombolytic agent. Exemplary thrombolytic agents include, but is not limited to, tissue plasminogen activator (t-PA), urokinase, anistreplase, ancrod, and brinase. It is demonstrated herein that the use of the composite molecule as an adjuvant therapy in combination with t-PA provides improved protection and decreased mortality compared to t-PA alone. Complement-targeting portion

In some embodiments, the complement-targeting portion of the herein described composition is CR2. The CR2 portion described herein comprises a CR2 or a fragment thereof. CR2 is a protein encoded by the CR2 gene and is involved in the complement system. It binds to iC3b (inactive derivative of C3b), C3dg, or C3d, B cells have CR2 receptors on their surfaces, allowing the complement system to play a role in B-cell activation and maturation. CR2 consists of an extracellular portion consisting of 15 or 16 repeating units known as short consensus repeats (SCRs). Amino acids 1-20 comprise the leader peptide, amino acids 23-82 comprise SCR1, amino acids 91-146 comprise SCR2, amino acids 154-210 comprise SCR3, amino acids 215-271 comprise SCR4. The active site (C3dg binding site) is located in SCR 1-2 (the first 2 N-terminal SCRs). SCR units are separated by short sequences of variable length that serve as spacers. It is understood that any number of SCRs containing the active site can be used. In one embodiment, the construct contains the 4 N-terminal SCR units. In another embodiment, the construct includes the first two N-terminal SCRs. In another

embodiment the construct includes the first three N-terminal SCRs.

In some embodiments, the CR2 portion comprises the first two N-terminal SCR domains of CR2. In some embodiments, the CR2 portion comprises the first three N-terminal SCR domains of CR2. In some embodiments, the CR2 portion comprises the first four N-terminal SCR domains of CR2. In some embodiments, the CR2 portion comprises (and in some embodiments consists of or consists essentially of) at least the first two N-terminal SCR domains of CR2, including for example at least any of the first 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 SCR domains of CR2.

A homologue of a CR2 protein or a fragment thereof includes proteins which differ from a naturally occurring CR2 (or CR2 fragment) in that at least one or a few amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derived (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol). In some embodiments, a CR2 homologue has an amino acid sequence that is at least about 70% identical to the amino acid sequence of a naturally occurring CR2 (e.g., SEQ ID NO: 1, or SEQ ID NO:2), for example at least about any of 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% identical to the amino acid sequence of a naturally occurring CR2 (e.g., SEQ ID NO: l, or SEQ ID NO:2). A CR2 homologue or a fragment thereof preferably retains the ability to bind to a naturally occurring ligand of CR2 (e.g., C3d or other C3 fragments with CR2 -binding ability). For example, the CR2 homologue (or fragment thereof) may have a binding affinity for C3d that is 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% of that of CR2 (or a fragment thereof) .

In some embodiments, the CR2 portion comprises at least the first two N- terminal SCR domains of a human CR2, such as a CR2 portion having an amino acid sequence containing at least amino acids 23 through 146 of the human CR2 (SEQ ID NO: 1). In some embodiments, the CR2 portion comprises at least the first two SCR domains of human CR2 having an amino acid sequence that is at least about any of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to amino acids 23 through 146 of the human CR2 (SEQ ID NO: l).

An amino acid sequence that is at least about, for example, 95% identical to a reference sequence (such as SEQ ID NO: 1 or SEQ ID NO: 2) is intended that the amino acid sequence is identical to the reference sequence except that the amino acid sequence may include up to five point alterations per each 100 amino acids of the reference sequence. These up to five point alterations may be deletions, substitutions, additions, and may occur anywhere in the sequence, interspersed either individually among amino acids in the reference sequence or in one or more continuous groups within the reference sequence.

In some embodiments, the CR2 portion comprises part or all of the ligand binding sites of the CR2 protein. In some embodiments, the CR2 portion further comprises sequences required to maintain the three dimensional structure of the binding site. Ligand binding sites of CR2 can be readily determined based on the crystal structures of CR2, such as the human and mouse CR2 crystal structures disclosed in U.S. Patent Application Publication No. 2004/0005538. For example, in some embodiments, the CR2 portion comprises the B strand and B-C loop of SCR2 of CR2. In some embodiments, the CR2 portion comprises a site on strand B and the B-C loop of CR2 SCR comprising the segment G98-G99-Y100-K101-I102-R103-G104-S105-T106-P107- Y108 with respect to SEQ ID NO: 1. In some embodiments, the CR2 portion comprises a site on the B strand of CR2 SCR2 comprising position Kl 19 with respect to SEQ ID NO: l . In some embodiments, the CR2 portion comprises a segment comprising V149- F150-P151-L152, with respect to SEQ ID NO: 1. In some embodiments, the CR2 portion comprises a segment of CR2 SCR2 comprising T120-N121-F122. In some embodiments, the CR2 -inhibitor molecule has two or more of these sites. For example, in some embodiments, the CR2 portion comprises a portion comprising G98-G99-Y100-K101- I102-R103-G104-5105-T106-P107-Y108 and Kl 19 with respect to SEQ ID NO: 1. Other combinations of these sites are also contemplated. Inhibitor Portion

The molecules described herein in some embodiments comprise an inhibitor portion comprising a complement modulator, such as a complement inhibitor.

As used herein, the term "complement inhibitor" refers to any compound, composition, or protein that reduces or eliminates complement activity. The reduction in complement activity may be incremental (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), or 90% reduction in activity) or complete. For example, in some embodiments, a complement inhibitor can inhibit complement activity by at least 10 (e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 or greater) % in a standard in vitro red blood cell hemolysis assay or an in vitro CH50eq assay. See, e.g., Kabat and Mayer (eds), "Experimental Immunochemistry, 2nd Edition," 135-240, Springfield, IL, CC Thomas (1961), pages 135-139, or a conventional variation of that assay such as the chicken erythrocyte hemolysis method as described in, e.g., Hillmen et al. (2004) N Engl J Med 350(6):552.

The CH50eq assay is a method for measuring the total classical complement activity in serum. This test is a lytic assay, which uses antibody- sensitized erythrocytes as the activator of the classical complement pathway and various dilutions of the test serum to determine the amount required to give 50% lysis (CH50). The percent hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of terminal complement complex (TCC) formation, since the TCC themselves are directly responsible for the hemolysis that is measured.

The assay is well known and commonly practiced by those of skill in the art. Briefly, to activate the classical complement pathway, undiluted serum samples (e.g., human serum samples) are added to microassay wells containing the antibody-sensitized erythrocytes to thereby generate TCC. Next, the activated sera are diluted in microassay wells, which are coated with a capture reagent (e.g., an antibody that binds to one or more components of the TCC). The TCC present in the activated samples bind to the monoclonal antibodies coating the surface of the microassay wells. The wells are washed and, to each well, is added a detection reagent that is detectably labeled and recognizes the bound TCC. The detectable label can be, e.g., a fluorescent label or an enzymatic label. The assay results are expressed in CH50 unit equivalents per milliliter (CH50 U Eq/mL).

Additional methods for detecting and/or measuring complement activity in vitro are set forth and exemplified in the working examples.

The complement inhibitor described herein in some embodiments is a specific inhibitor of the lectin pathway. In some embodiments, the complement inhibitor is a specific inhibitor of the alternative pathway. In some embodiments, the complement inhibitor is a specific inhibitor of the classical pathway.

In some embodiments, the complement inhibitor is a soluble or membrane-bound protein such as, for example, membrane cofactor protein (MCP), decay accelerating factor (DAF/CD55), CD59, mouse complement receptor 1-related gene/protein y (Crry), human complement receptor 1 (CR1) or factor H, or Factor I, or an antibody specific for a component of a complement pathway such as, for example, eculizumab (an anti-CS antibody marketed under the trade name Soliris®), pexelizumab (the antigen-binding fragment of eculizumab), an anti-factor B antibody (such as the monoclonal antibody 1379 produced by ATCC Deposit No. PTA-6230), an anti- properdin antibody, an anti-factor D antibody, an anti-MASP antibody, an anti MB L- antibody, and the like (see below). Alternatively, a complement inhibitor may be a small molecule or a linear or cyclic peptide such as, for example, compstatin, N- acetylaspartylglutamic acid (NAAGA), and the like. In some embodiments, the complement inhibitor is selected from the group consisting of: an anti-C5 antibody, an Eculizumab, an pexelizumab, an anti-C3b antibody, an anti-C6 antibody, an anti-C7 antibody, an anti- factor B antibody, an anti- factor D antibody, and an anti-properdin antibody, a human membrane co factor protein (MCP), a human decay accelerating factor (DAF), a mouse decay accelerating factor (DAF), a human CD59, a mouse CD59, a mouse CD59 isoform B, a mouse Crry, a human CR1, a Factor I, a human factor H, a mouse factor H, and a biologically active fragment of any the preceding.

As used herein, the term "membrane cofactor protein," "MCP," or "CD46" refers to a widely distributed C3b/C4b-binding cell surface glycoprotein which inhibits complement activation on host cells and serves as a cofactor for the factor I-mediated cleavage of C3b and C4b, including homologs thereof. T.J. Oglesby et al., J. Exp. Med. (1992) 175: 1547-1551. MCP belongs to a family known as the regulators of complement activation ("RCA"). Family members share certain structural features, comprising varying numbers of short consensus repeat (SCR) domains, which are typically between 60 and 70 amino acids in length. Beginning at its amino-terminus, MCP comprises four SCRs, a serine/threonine/proline-enriched region, an area of undefined function, a transmembrane hydrophobic domain, a cytoplasmic anchor and a cytoplasmic tail. It is understood that species and strain variations exist for the disclosed peptides,

polypeptides, and proteins, and that human MCP or biologically active fragments thereof encompass all species and strain variations.

SEQ ID NO: 3 represents the full-length human MCP amino acid sequence (see, e.g., UniProtKB/Swiss-Prot. Accession No. P15529). Amino acids 1-34 correspond to the signal peptide, amino acids 35-343 correspond to the extracellular domain, amino acids 344-366 correspond to the transmembrane domain, and amino acids 367-392 correspond to the cytoplasmic domain. In the extracellular domain, amino acids 35-96 correspond to SCR 1, amino acids 97-159 correspond to SCR 2, amino acids 160-225 correspond to SCR 3, amino acids 226-285 correspond to SCR 4, and amino acids 302- 326 correspond to the serine/threonine-rich domain. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that MCP or biologically active fragments thereof encompass all species and strain variations. As used herein, the term "biologically active" fragment of MCP refers to any soluble fragment lacking both the cytoplasmic domain and the transmembrane domain, including fragments comprising, consisting essentially of or consisting of 1, 2, 3, or 4 SCR domains, with or without the serine/threonine-rich domain, having some or all of the complement inhibitory activity of the full-length human MCP protein. In some embodiments, the complement inhibitor portion comprises full-length human MCP (amino acids 35-392 of SEQ ID NO:3), the extracellular domain of human MCP (amino acids 35-343 of SEQ ID NO:3), or SCRs 1-4 of human MCP (amino acids 35-285 of SEQ ID NO:3).

Decay accelerating factor, also referred to as CD55 (DAF/CD55) (SEQ ID NO:4 and SEQ ID NO:5), is an -70 kiloDalton (kDa) membrane-bound glycoprotein which inhibits complement activation on host cells. Like several other complement regulatory proteins, DAF comprises several approximately 60 amino acid repeating motifs termed short consensus repeats (SCR).

As used herein, the term "decay accelerating factor," "DAF," or "CD55" refers to a seventy kilodalton ("kDa") membrane glycoprotein comprising four short consensus repeat (SCR) domains followed by a heavily O-glycosylated serine/threonine- rich domain at the C-terminus that elevates the molecule from the membrane surface, followed by a glycosylphosphatidylinositol ("GPI") anchor. DAF protects the cell surface from complement activation by dissociating membrane-bound C3 convertases that are required to cleave complement protein C3 and to amplify the complement cascade. DAF prevents assembly or accelerates decay of both the C3- and C5-convertases of the alternative and classical complement pathways.

SEQ ID NO:4 represents the full-length human DAF amino acid sequence

(see, e.g., UniProtKB/Swiss-Prot. Accession No. P08173); SEQ ID NO:5 represents the full-length mouse DAF amino acid sequence (see, e.g., UniProtKB/Swiss-Prot. Accession No. Q61475). In the human DAF sequence, amino acids 1-34 correspond to the signal peptide, amino acids 35-353 appear in the mature protein, and amino acids 354-381 are removed from the polypeptide after translation. Within the mature protein, amino acids 35-96 correspond to SCR 1, amino acids 96- 160 correspond to SCR 2, amino acids 161- 222 correspond to SCR 3, amino acids 223-285 correspond to SCR 4, and amino acids 287-353 correspond to the O-glycosylated serine/threonine-rich domain. The GPI anchor is attached to human DAF at a serine at position 353. In the mouse DAF sequence, amino acids 1-34 correspond to the signal peptide, amino acids 35-362 appear in the mature protein, and amino acids 363-390 are removed from the polypeptide after translation. Within the mature protein, amino acids 35-96 correspond to SCR 1, amino acids 97-160 correspond to SCR 2, amino acids 161-222 correspond to SCR 3, amino acids 223-286 correspond to SCR 4, and amino acids 288-362 correspond to the O-glycosylated serine/threonine-rich domain. The GPI anchor is attached to mouse DAF at a serine at position 362. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that DAF or biologically active fragments thereof encompass all species and strain variations. As used herein, the term "biologically active" fragment of DAF refers to any fragment of DAF lacking a GPI anchor and/or the amino acid to which it is attached (i.e., Ser-353), including any fragments of the full- length DAF protein comprising, consisting essentially of or consisting of 1, 2, 3, or 4 SCR domains, with or without the O-glycosylated serine/threonine-rich domain, having some or all the complement inhibitory activity of the full-length DAF protein.

As used herein, the term "CD59" refers to a membrane-bound 128 amino acid glycoprotein that potently inhibits the membrane attack complex (MAC) of complement. CD59 acts by binding to the C8 and/or C9 components of the MAC during assembly, ultimately preventing incorporation of the multiple copies of C9 required for complete formation of the osmolytic pore at the heart of the MAC. CD59 is both N- and O-glycosylated. The N-glycosylation comprises primarily bi- or tri-antennary structures with and without lactosamine and outer arm fucose residues, with variable sialylation present at some sites. Like DAF, CD59 is anchored in the cell membrane by a glycosylphosphatidylinositol ("GPI") anchor, which is attached to an asparagine at amino acid 102. Soluble forms of CD59 (sCD59) have been produced, but they generally have low functional activity in vitro, particularly in the presence of serum, suggesting that unmodified sCD59 has little or no therapeutic efficacy. See, e.g., S. Meri et al.,

"Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser- desorption mass spectrometric analysis," Biochem. J. 316:923-935 (1996).

SEQ ID NO:6 represents the full-length human CD59 amino acid sequence (see, e.g., UniProtKB/Swiss-Prot. Accession No. P13987); SEQ ID NO:7 represents the full-length mouse CD59 sequence, isoform A (see, e.g., UniProtKB/Swiss- Prot. Accession No. 055186); SEQ ID NO:8 represents the full-length mouse CD59 sequence, isoform B (see, e.g., UniProtKB/SwissProt. Accession No. P58019). In the human CD59 sequence, amino acids 1-25 of SEQ ID NO:6 correspond to the leader peptide, amino acids 26-102 of SEQ ID NO: 6 correspond to the mature protein, and amino acids 103-128 of SEQ ID NO:6 are removed after translation. The GPI anchor is attached to CD59 at an asparagine at position 102 of SEQ ID NO:6. In isoform A of the mouse CD59 sequence, amino acids 1-23 of SEQ ID NO:7 correspond to the leader peptide, amino acids 24-96 of SEQ ID NO: 7 correspond to the mature protein, and amino acids 97-123 of SEQ ID NO:7 are removed after translation. The GPI anchor is attached to CD59 at a serine at position 96 of SEQ ID NO: 7. In isoform B of the mouse CD59 sequence, amino acids 1-23 of SEQ ID NO: 8 correspond to the leader peptide, amino acids 24-104 of SEQ ID NO: 8 correspond to the mature protein, and amino acids 105-129 of SEQ ID NO:8 are removed after translation. The GPI anchor is attached to CD59 at an asparagine at position 104 of SEQ ID NO:8. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that CD59 or biologically active fragments thereof encompass all species and strain variations.

As used herein, the term "biologically active" fragment of human CD59 refers to any fragment of human CD59 lacking a GPI anchor and/or the amino acid to which it is attached (i.e., Asn-102), including any fragments of the full-length human CD59 protein having some or all the complement inhibitory activity of the full-length CD59 protein; and the term "biologically active" fragment of mouse CD59 refers to any fragment of mouse CD59 isoform A or isoform B lacking a GPI anchor and/or the amino acid to which it is attached (i.e., Ser-96 of isoform A, or Asp- 104 of isoform B), including any fragments of either full-length mouse CD59 protein isoform having some or all the complement inhibitory activity of the full-length CD59 protein. As used herein, the term "mouse complement receptor 1 -related gene/protein y" or "Crry" refers to a membrane-bound mouse glycoprotein that regulates complement activation, including homologs thereof. Crry regulates complement activation by serving as a cofactor for complement factor I, a serine protease which cleaves C3b and C4b deposited on host tissue. Crry also acts as a decay- accelerating factor, preventing the formation of C4b2a and C3bBb, the amplification convertases of the complement cascade.

SEQ ID NO: 9 represents the full-length mouse Crry protein amino acid sequence. Amino acids 1-40 correspond to the leader peptide, amino acids 41-483 of SEQ ID NO:9 correspond to the mature protein, comprising amino acids 41-405 of SEQ ID NO: 9, corresponding to the extracellular domain, amino acids 406-426 of SEQ ID NO: 9, corresponding to the transmembrane domain, and amino acids 427-483 of SEQ ID NO: 9, corresponding to the cytoplasmic domain. In the extracellular domain, amino acids 83-143 of SEQ ID NO: 9 correspond to SCR 1, amino acids 144-205 of SEQ ID NO: 9 correspond to SCR 2, amino acids 206-276 of SEQ ID NO: 9 correspond to SCR 3, amino acids 277-338 of SEQ ID NO: 9 correspond to SCR 4, and amino acids 339-400 of SEQ ID NO:9 correspond to SCR 5. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that mouse Crry protein or biologically active fragments thereof encompasses all species and strain variations. As used herein, the term "biologically active" fragment of mouse Crry protein refers to any soluble fragment of mouse Crry lacking the transmembrane domain and the cytoplasmic domain, including fragments comprising, consisting essentially of or consisting of 1, 2, 3, 4, or 5 SCR domains, including any fragments of the full-length mouse Crry protein having some or all the complement inhibitory activity of the full-length Crry protein. In one embodiment, the biologically active fragment of mouse Crry comprises amino acids 85-403 of SEQ ID NO: 9.

As used herein, the term "complement receptor 1," "CR1 ," or "CD35" refers to a human gene encoding a protein of 2039 amino acids, with a predicted molecular weight of 220 kilodaltons ("kDa"), including homologs thereof. The gene is expressed principally on erythrocytes, monocytes, neutrophils, and B cells, but is also present on some T lymphocytes, mast cells, and glomerular podocytes. CR1 protein is typically expressed at between 100 and 1000 copies per cell. CR1 is the main system for processing and clearance of complement-opsonized immune complexes. CR1 negatively regulates the complement cascade, mediates immune adherence and phagocytosis, and inhibits both the classic and alternative complement pathways. The full-length CR1 protein comprises a 42 amino acid signal peptide, an extracellular domain of 1930 amino acids, a 25 amino acid transmembrane domain, and a 43 amino acid C-terminal cytoplasmic domain. The extracellular domain of CR1 has 25 potential N-glycosylation signal sequences, and comprises 30 short consensus ("SCR") domains, also known as complement control protein (CCP) repeats, or sushi domains, each 60 to 70 amino acids long. The sequence homology between SCRs ranges between 60-99 percent. The 30 SCR domains are further grouped into four longer regions termed long homologous repeats ("LHRs"), each encoding approximately 45 kDa segments of the CR1 protein, designated LHR-A, -B, -C, and -D. The first three comprise seven SCR domains each, while LHR-D comprises 9 SCR domains. The active sites on the extracellular domain of CR1 protein include a C4b-binding site with lower affinity for C3b in SCRs 1-4 comprising amino acids 42-295, a C3b-binding site with lower affinity for C4b in SCRs 8-11 comprising amino acids 490-745, a C3b-binding site with lower affinity for C4b in SCRs 15-18 comprising amino acids 940-1196, and a Clq-binding site in SCRs 22-28 comprising amino acids 1394-1842.

SEQ ID NO: 10 represents the full-length human CR1 amino acid sequence

(see, e.g., UniProtKB/Swiss-Prot. Accession No. P17927). Amino acids 1-41 correspond to the signal peptide, amino acids 42-2039 correspond to the mature protein, comprising amino acids 42-1971, corresponding to the extracellular domain, amino acids 1972-1996, corresponding to the transmembrane domain, and amino acids 1997-2039, corresponding to the cytoplasmic domain. In the extracellular domain, amino acids 42-101 correspond to SCR 1, 102-163 correspond to SCR2, amino acids 164-234 correspond to SCR3, amino acids 236-295 correspond to SCR4, amino acids 295-355 correspond to SCR5, amino acids 356-418 correspond to SCR6, amino acids 419-489 correspond to SCR7, amino acids 491-551 correspond to SCR8, amino acids 552- 613 correspond to SCR9, amino acids 614-684 correspond to SCRIO, amino acids 686-745 correspond to SCR1 1, amino acids 745-805 correspond to SCR12, amino acids 806-868 correspond to SCR13, amino acids 869-939 correspond to SCR 14, amino acids 941-1001 correspond to SCR15, amino acids 1002-1063 correspond to SCR16, amino acids 1064-1134 correspond to SCR17, amino acids 1136-1195 correspond to SCR18, amino acids 1195-1255 correspond to SCR 19, amino acids 1256-1318 correspond to SCR 20, amino acids 1319-1389 correspond to SCR 21, amino acids 1394-1454 correspond to SCR 22, amino acids 1455-1516 correspond to SCR 23, amino acids 1517-1587 correspond to SCR 24, amino acids 1589- 1648 correspond to SCR 25, amino acids 1648-1708 correspond to SCR 26, amino acids 1709-1771 correspond to SCR 27, amino acids 1772-1842 correspond to SCR 28, amino acids 1846-1906 correspond to SCR 29, amino acids 1907-1967 correspond to SCR 30. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that CR1 protein or biologically active fragments thereof encompass all species and strain variations. As used herein, the term "biologically active" fragment of CR1 protein refers to any soluble fragment of CR1 lacking the

transmembrane domain and the cytoplasmic domain, including fragments comprising, consisting essentially of or consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 SCR domains, including any fragments of the full-length CR1 protein having some or all the complement inhibitory activity of the full-length CR1 protein.

As used herein, the term "complement factor H," "factor H," or "FH" refers to complement factor H, a single polypeptide chain plasma glycoprotein, including homologs thereof. The protein is composed of 20 conserved short consensus repeat (SCR) domains of approximately 60 amino acids, arranged in a continuous fashion like a string of beads, separated by short linker sequences of 2-6 amino acids each. Factor H binds to C3b, accelerates the decay of the alternative pathway C3-convertase (C3bBb), and acts as a cofactor for the proteolytic inactivation of C3b. In the presence of factor H, proteolysis by factor I results in the cleavage and inactivation of C3b. Factor H has at least three distinct binding domains for C3b, which are located within SCRs 1-4, SCRs 5- 8, and SCRs 19-20. Each domain binds to a distinct region within the C3b protein: the N- terminal sites bind to native C3b; the second site, located in the middle region of factor H, binds to the C3c fragment and the site located within SCR19 and 20 binds to the C3d region. In addition, factor H also contains binding sites for heparin, which are located within SCR 7, SCRs 5-12, and SCR 20 of factor Hand overlap with those of the C3b binding sites. Structural and functional analyses have shown that the domains for the complement inhibitory activity of factor H are located within the first four N-terminal SCR domains.

SEQ ID NO: 11 represents the full-length human factor H amino acid sequence (see, e.g., UniProtKB/Swiss-Prot. Accession No. P08603); SEQ ID NO: 12 represents the full-length mouse factor H amino acid sequence (see, e.g.,

UniProtKB/Swiss-Prot. Accession No. P06909). In the human factor H sequence, amino acids 1-18 of SEQ ID NO: 11 correspond to the signal peptide, and amino acids 19-1231 of SEQ ID NO: 11 correspond to the mature protein. Within that protein, amino acids 21- 80 of SEQ ID NO: 11 correspond to SCR 1, amino acids 85-141 of SEQ ID NO:52 correspond to SCR 2, amino acids 146-205 of SEQ ID NO: 11 correspond to SCR 3, amino acids 210-262 of SEQ ID NO: 11 correspond to SCR 4, and amino acids 267-320 of SEQ ID NO: l 1 correspond to SCR 5. In the mouse factor H sequence, amino acids 1- 18 of SEQ ID NO: 12 correspond to the signal peptide, and amino acids 19-1234 of SEQ ID NO: 12 correspond to the mature protein. Within that protein, amino acids 19-82 of SEQ ID NO: 12 correspond to SCR 1, amino acids 83-143 of SEQ ID NO: 12 correspond to SCR 2, amino acids 144-207 of SEQ ID NO: 12 correspond to SCR 3, amino acids 208-264 of SEQ ID NO: 12 correspond to SCR 4, and amino acids 265-322 of SEQ ID NO: 12 correspond to SCR 5. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that factor H or biologically active fragments thereof encompass all species and strain variations.

As used herein, the term "biologically active" fragment of factor H refers to any portion of a factor H protein having some or all the complement inhibitory activity of the full-length factor H protein, and includes, but is not limited to, factor H fragments comprising SCRs 1-4, SCRs 1-5, SCRs 1-8, SCRs 1-18, SCRs 19-20, or any homolog of a naturally-occurring factor H or fragment thereof, as described in detail below. In some embodiments, the biologically active fragment of factor H has one or more of the following properties: (1) binding to C-reactive protein (CRP), (2) binding to C3b, (3) binding to heparin, (4) binding to sialic acid, (5) binding to endothelial cell surfaces, (6) binding to cellular integrin receptor, (7) binding to pathogens, (8) C3b co-factor activity, (9) C3b decay- acceleration activity, and (10) inhibiting the alternative complement pathway.

SEQ ID NO: 13 represents the amino acid sequence for mannose-binding lectin-associated protein of 44kDa (MAp44). MAp44 is an alternatively spliced product encoded by the MASP1 gene. In certain aspects, MAp44 is an inhibitor of lectin pathway activation.

Thus, in some embodiments, the inhibitor portion of the targeted molecule described herein comprises a complement inhibitor or biologically active fragment thereof. In some embodiments, the complement inhibitor is selected from the group consisting of human MCP, human DAF, mouse DAF, human CD59, mouse CD59 isoform A, mouse CD59 isoform B, mouse Crry protein, human CRl, human factor H, or mouse factor H, a Factor I, MAp44 or a biologically active fragment thereof.

In some embodiments, the inhibitor portion comprises full-length human MCP (SEQ ID NO:3). In some embodiments, the complement inhibitor portion of the targeting construct comprises a biologically active fragment of human MCP (SEQ ID

NO:3). In some embodiments, the biologically active fragment of human MCP is selected from the group consisting of SCRs 1-4 (amino acids 35-285 of SEQ ID NO:3), SCRs 1-4 plus the serine/threonine-rich domain (amino acids 35-326 of SEQ ID NO:3), and the extracellular domain of MCP (amino acids 35-343 of SEQ ID NO:3).'

In some embodiments, the inhibitor portion comprises full-length human

DAF. In some embodiments, the inhibitor portion comprises a biologically active fragment of human DAF (SEQ ID NO: 4). In some embodiments, the biologically active fragment of human DAF is selected from the group consisting of SCRs 1-4 (amino acids 25-285 of SEQ ID NO:4) and SCRs 1-4 plus the O- glycosylated serine/threonine-rich domain (amino acids 25-353 of SEQ ID NO:4). In some embodiments, the inhibitor portion comprises full-length mouse DAF (SEQ ID NO:5). In some embodiments, the inhibitor portion comprises a biologically active fragment of mouse DAF. In some embodiments, the biologically active fragment of mouse DAF is selected from the group consisting of SCRs 1-4 (amino acids 35-286 of SEQ ID NO:5) and SCRs 1-4 plus the O- glycosylated serine/threonine-rich domain (amino acids 35-362 of SEQ ID NO:5). In some embodiments, the inhibitor portion comprises full-length human CD59 (SEQ ID NO:6). In some embodiments, the inhibitor portion comprises a biologically active fragment of human CD59 (SEQ ID NO:6). In some embodiments, the biologically active fragment of human CD59 comprises the extracellular domain of human CD59 lacking its GPI anchor (amino acids 26-101 of SEQ ID NO:6). In some embodiments, the inhibitor portion comprises full-length mouse CD59, isoform A (SEQ ID NO:6). In some embodiments, the inhibitor portion comprises a biologically active fragment of mouse CD59, isoform A (SEQ ID NO:7). In some embodiments, the biologically active fragment of mouse CD59, isoform A comprises the extracellular domain of mouse CD59, isoform A lacking its GPI anchor (amino acids 24-95 of SEQ ID NO: 7). In some embodiments, the inhibitor portion comprises full-length mouse CD59, isoform B (SEQ ID NO:8). In some embodiments, the c inhibitor portion comprises a biologically active fragment of mouse CD59, isoform B (SEQ ID NO:8). In some embodiments, the biologically active fragment of mouse CD59, isoform B comprises the extracellular domain of mouse CD59, isoform lacking its GPI anchor (amino acids 24- 103 of SEQ ID NO: 8).

In some embodiments, the inhibitor portion comprises full-length mouse Crry protein (SEQ ID NO:9). In some embodiments, the inhibitor portion comprises a biologically active fragment of mouse Crry protein (SEQ ID NO:9). In some

embodiments, the biologically active fragment of mouse Crry protein is selected from the group consisting of SCRs 1-5 (amino acids 41-400 of SEQ ID NO:9) and the

extracellular domain of mouse Crry protein (amino acids 41-405 of SEQ ID NO:9). In one embodiment, the inhibitor portion comprises the biologically active fragment of mouse Crry comprising amino acids 85-403 of SEQ ID NO: 9.

In some embodiments, the inhibitor portion comprises full-length human

CRl protein (SEQ ID NO: 10). In some embodiments, the t inhibitor portion comprises a biologically active fragment of human CRl protein (SEQ ID NO: 10). In some embodiments, the biologically active fragment of human CRl protein is selected from the group consisting of SCRs 1-4 (amino acids 42-295 of SEQ ID NO: 10), SCRs 1-10 (amino acids 42-684 of SEQ ID NO: 10), SCRs 8-11 (amino acids 490-745 of SEQ ID NO: 10), SCRs 15-18 (amino acids 940-1196 of SEQ ID NO: 10), and SCRs 22-28 (amino acids 1394-1842 of SEQ ID NO: 10).

In some embodiments, the inhibitor portion comprises full-length human (SEQ ID NO: 11) or mouse (SEQ ID NO: 12) factor H. In some embodiments, the inhibitor portion comprises a biologically active fragment of human (SEQ ID NO: l 1) or mouse (SEQ ID NO: 12) factor H. In some embodiments, the biologically active fragment of human factor H (SEQ ID NO: 11) is selected from the group consisting of SCRs 1-4 (amino acids 21-262 of SEQ ID NO: 11), SCRs 1-5 of factor H (amino acids 21-320 of SEQ ID NO: 11), SCRs 1-8 of factor H (amino acids 21-507 of SEQ ID NO: 11), and SCRs 1-18 of factor H (amino acids 21-1104 of SEQ ID NO: 11). In some embodiments, the biologically active fragment of mouse factor H (SEQ ID NO: 12) is selected from the group consisting of SCRs 1-4 (amino acids 19-264 of SEQ ID NO: 12), SCRs 1-5 of factor H (amino acids 19-322 of SEQ ID NO: 12), SCRs 1-8 of factor H (amino acids 19- 507 of SEQ ID NO: 12), and SCRs 1-18 of factor H (amino acids 19-1109 of SEQ ID NO: 12). In some embodiments, the biologically active fragment of human (SEQ ID NO: 11) or mouse (SEQ ID NO: 12) factor H comprises (and in some embodiments consists of or consists essentially of) at least the first four N-terminal SCR domains of factor H, including for example, at least any of the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more N-terminal SCR domains of factor H.

In some embodiments, the inhibitor portion comprises MAp44 (SEQ ID

NO: 13). In some embodiments, the inhibitor portion comprises a biologically active fragment of MAp44 (SEQ ID NO: 13).

In some embodiments, the inhibitor portion of the targeted molecules is a homolog of any of the complement inhibitors described herein or a biologically active fragment thereof. Homologs of the complement inhibitors (or biologically active fragments thereof) include proteins which differ from a naturally occurring complement inhibitor (or biologically-active fragment thereof) in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition glycosylphosphatidyl inositol). For example, homologue of a complement inhibitor may have an amino acid sequence that is at least about 70% identical to the amino acid sequence of a naturally complement inhibitor (e.g., SEQ ID NOs:44-53), for example at least about any of 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% identical to the amino acid sequence of a naturally occurring complement inhibitor (e.g., SEQ ID NOs: 3-13). Amino acid sequence identity can be determined in various ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNAST AR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

In certain embodiments, a homolog of complement inhibitor (or a biologically active fragment thereof) retains all the alternative complement pathway inhibitory activity of the complement inhibitor (or a biologically active fragment thereof) from which it is derived. In certain embodiments, the homolog of a complement inhibitor (or a biologically-active fragment thereof) retains at least about 50%, for example, at least about any of 60%, 70%, 80%, 90%, or 95% of the complement inhibition activity the complement inhibitor (or a biologically-active fragment thereof) from which is derived.

In some embodiments, the inhibitor portion comprises an antibody (or an antigen binding fragment thereof) that binds to a complement component, e.g., a complement component selected from the group consisting of CI, Clq, Cis, C2, C2a, C3, C3a, C3b, C4, C4b, C5, C5a, C5b, C6, C7, C8, and C9. The complement polypeptides to which the antibodies or antigen binding fragments thereof bind can be, in some embodiments, human polypeptides, e.g., human CI, Clq, Cls, C2, C2a, C3, C3a, C3b, C4, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, or properdin polypeptides. The amino acid sequences for the foregoing complement proteins are well-known in the art as are methods for preparing the proteins or fragments thereof for use in preparing an antibody (or antigen-binding fragment thereof) specific for one or more of the

complement proteins. Suitable methods are also described and exemplified herein. Exemplary anti-complement protein antibodies, which are suitable for incorporation into the targeted molecules described herein and for subsequent use in any of the methods described herein, are also well known in the art. For example, antibodies that bind to complement component C5 and inhibit the cleavage of C5 into fragments C5a and C5b include, e.g., eculizumab (Soliris®; Alexion Pharmaceuticals, Inc.,

Cheshire, CT) and pexelizumab (Alexion Pharmaceuticals, Inc., Cheshire, CT). See, e.g., Kaplan (2002) Curr Opin Investig Drugs 3(7): 1017-23; Hill (2005) Clin Adv Hematol Oncol 3(11):849-50; Rother et al. (2007) Nature Biotechnol 25(11): 1256-1488; Whiss (2002) Curr Opin Investig Drugs 3(6):870-7; Patel et al. (2005) Drugs Today (Bare) 41(3): 165-70; and Thomas et al. (1996) Mol Immunol. 33(17- 18): 1389-401.

In some embodiments, the anti-C5 antibody can bind to an epitope in the alpha chain of the human complement component C5 protein. Antibodies that bind to the alpha chain of C5 are described in, for example, PCT application publication no. WO 2010/136311 and U.S. patent no. 6,355,245. In some embodiments, the anti-C5 antibody can bind to an epitope in the beta chain of the human complement component C5 protein. Antibodies that bind to the C5 beta chain are described in, e.g., Moongkamdi et al. (1982) Immunobiol 162:397; Moongkamdi et al. (1983) Immunobiol 165:323; and Mollnes et al. (1988) Scand 1 Immunol 28:307-312.

Additional anti-C5 antibodies, and antigen-binding fragments thereof, suitable for use in the targeting constructs described herein are described in, e.g., PCT application publication no. WO 2010/015608, the disclosure of which is incorporated herein by reference in its entirety.

Antibodies that bind to C3b and, for example, inhibit the C3b convertase are also well known in the art. For example, PCT application publication nos. WO 2010/136311, WOb2009/056631, and WO 2008/154251, the disclosures of each of which are incorporated herein by reference in their entirety. Antagonistic anti-C6 antibodies and anti-C7 antibodies have been described in, e.g., Brauer et al. (1996) Transplantation 61(4):S88-S94 and U.S. patent no. 5,679,345.

In some embodiments, the inhibitor portion comprises an anti-factor B antibody (such as the monoclonal antibody 1379 produced by ATCC Deposit No. PTA- 6230). Anti-factor B antibodies are also described in, e.g., Ueda et al. (1987) J Immunol 138(4): 1143-9; Tanhehco et al. (1999) Transplant Proc 31(5):2168-71; U.S. patent application publication nos. 20050260198 and 2008029911; and PCT publication no. WO 09/029669.

In some embodiments, the inhibitor portion comprises an anti-factor D antibody, e.g., an antibody described in Pascual et al. (1990) 1 Immunol Methods

127:263-269; Sahu et al. (1993) Mol Immunol 30(7):679-684; Pascual et al. (1993) Eur 1 Immunol 23 : 1389-1392; Niemann et al. (1984) J Immunol 132(2):809-815; U.S. patent no. 7,439,331; or U.S. patent application publication no. 20080118506.

In some embodiments, the inhibitor portion comprises an anti-properdin antibody. Suitable anti-properdin antibodies are also well-known in the art and include, e.g., U.S. patent application publication nos. 20110014614 and PCT application publication no. W02009110918.

In some embodiments, the inhibitor portion comprises an anti-MBL antibody. Mannose-binding mannan-binding lectin (MBL), a plasma protein, forms a complex with proteins known as MBL-associated serine proteases (MASPs). MBL binds to several monosaccharides that are uncharacteristic of mammalian proteins, e.g., mannose, N-acetylglucosamine, N-acetylmannoseamine, L-fucose and glucose, whereas sialic acid and galactose are not bound. When the MBL-MASP complex binds to microorganisms, the proenzymic forms of the serine proteases are activated and mediate the activation of complement components C4 and C2, thereby generating the C3 convertase C4b2b and leading to opsonization by the deposition of C4b and C3b fragments. MASP -2 has been shown to cleave C4 and C2, while MASP-1 may be responsible for direct cleavage of C3. The functions of MASP-3 and MApl9 are less well understood. Studies have shown a clear link between low levels of MBL and opsonic deficiency, as well as clinical manifestations such as severe diarrhea, chronic hepatitis and HIV infection, and autoimmune disease. See, Petersen et al., J. Immunological Methods, 257: 107-16 (2001); Petersen et al., Molecular Immunology, 38: 133-49 (2001). Anti-mannan-binding lectin antibodies are known in the art (see, e.g., Pradhan et al.

(2012) Rheumatol. Int. epublished September, 2012) and commercially available

(AbCam). In some embodiments, the inhibitor portion comprises an anti-MASP antibody. The mannan-binding lectin-associated serine proteases (MASPs) are a family of at least three proteins (mannan-binding lectin-associated serine protease- 1, -2 and -3 (MASP-1, MASP-2 and MASP-3, respectively)), which have been taught to play a significant role in modulation of the lectin pathway of complement activation. Petersen et al., Molecular Immunology 38: 133-149 (2001).

MASP-1 has a histidine loop structure of the type found in trypsin and trypsin-like serine proteases. MASP-1 has been found to be involved in complement activation by MBL. A cDNA clone encoding MASP-1 has been reported that encodes a putative leader peptide of 19 amino acids followed by 680 amino acid residues predicted to form the mature peptide. MASP-2 (MBL-associated serine protease 2) is a serine protease also similar in structure to CI r and CI s of the complement pathway. Like these, and contrary to MASP-1, it has no histidine loop structure of the type found in trypsin and trypsin-like serine proteases. It has been theorized that MASP-1 can cleave C3, generating C3b, which may be deposited on an activated cell or tissue surface

It has been shown that MASP-2, cleaves C4 and C2, giving rise to the C3 convertase, C4b2b (Thiel et al., Nature, 386:506-10 (1997)). The MASP-2 protein comprises of a number of domains namely the CUB1, EGF, CUB2, CCP1, CCP2 and serine protease domains. It is believed that the domain responsible for association with MBL is situated in the N-terminus, whereas the serine protease domain is responsible for the serine protease activity of MASP-2. sMAP, also known as MApl9, is a 19 kd is derived from the same gene as MASP-2, which lacks the serine protease domain and a major part of the A chain. Skjoedt et al., Immunobiology, 215:921-31 (2010). Recently, a third member of the family, MASP-3 was identified, which shares a high degree of homology with MASP-1, such that it appears that MASP-1 and MASP-3 are generated as a result of alternative splicing of primary mRNA transcripts.

Antibodies against MBL, MASP- 1, MASP-2, MASP-3 and the

MBL/MASP complex, and their use for inhibiting the adverse effects of complement activation, such as ischemia-reperfusion injury, have been disclosed, for example, in WO04/075837; US 2009/0017031. Other antibodies to MASP-2 have been described previously, as well. See, e.g., WO 02/06460, US2007/0009528, Peterson et al., Mol. Immunol. 37:803-11 (2000), MoUer-Kristensen et al., J. of Immunol. Methods 282: 159-67 (2003), Petersen et al., Mol. Immunol. 35:409, and WO 04/106384.

An additional related protein, MBL/Ficolin Associated Protein (MAP- 1), which is present in low serum levels compared to MASP-1 and MASP-3, has been reported to function as a local lectin pathway specific complement inhibitor. Skjodt et al., Molecular Immunology, 47:2229-30 (2010). Accordingly, MAP-1 itself, or fragments of MAP-1, may be useful in the present invention as an inhibitor of MASP, and accordingly, as a lectin-pathway- specific inhibitor of complement activation. Finally, the ficolin family of proteins are characterized by carbohydrate binding and opsonic activities, sharing a structure similar to MBL. Like MBL, the ficolins have been shown to associate with MASPs in serum and may mediate complement activation in response to

pathogenic, necrotic, or apoptotic cell- specific carbohydrate markers. Accodingly, inhibitors of the ficolin family or functional fragments therof may be useful in the present invention as an inhibtor of MASPs and as a lectin-pathway specific inhibitor of complement activation. US Patent 6,333,034 and US Patent 7,423,128; see also, WO 2008/154018 and WO 2009/110918.

In some embodiments, the inhibitor portion comprises an antibody (or antigen binding fragment thereof) that specifically binds to a human complement component protein (e.g., human C5, C6, C7, C8, or C9). The terms "specific binding" or "specifically binds" refer to two molecules forming a complex (e.g., a complex between an antibody and a complement component protein) that is relatively stable under physiologic conditions. Typically, binding is considered specific when the association constant (Ka) is higher than 106 M-l. Thus, an antibody can specifically bind to a C5 protein with a Ka of at least (or greater than) 106 (e.g., at least or greater than 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015 or higher) M-l. Examples of antibodies that specifically bind to a human complement component C5 protein are described in, e.g., U.S. patent no. 6,355,245 and PCT application publication no. WO 2010/015608.

Methods for determining whether an antibody binds to a protein antigen and/or the affinity for an antibody to a protein antigen are known in the art and described herein. For example, the binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.), or enzyme-linked immunosorbent assay (ELISA) assays. See, e.g., Harlow and Lane (1988) "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Benny K. C. Lo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," W.H. Freeman and Co., NY; Borrebaek (1995) "Antibody Engineering," 2nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993) 1 Immunol Meth. 160: 191- 198; Jonsson et al. (1993) Ann Biol Clin 51 : 19-26; and Jonsson et al. (1991) Biotechniques 11 :620-627. See also, U.S. Patent No. 6,355,245.

In any of the embodiments described herein, the targeted molecule also includes an amino acid linker sequence linking the targeting portion and the inhibitor portion.

Composite Molecule

In some embodiments, the CR2 portion and the inhibitor portion are non- covalently linked. For example, the two portions may be brought together by two interacting bridging proteins (such as biotin and streptavidin), each linked to a CR2 portion or the inhibitor portion.

In one embodiment, the molecule described herein thus generally has the dual functions of binding to a CR2 ligand and inhibiting complement activation of the alternative pathway. "CR2 ligand" refers to any molecule that binds to a naturally occurring CR2 protein, which include, but are not limited to, C3d, iC3b, C3dg, C3d, and cell-bound fragments of C3b that bind to the two N-terminal SCR domains of CR2. The CR2 -inhibitor molecule may, for example, bind to a CR2 ligand with a binding affinity that is about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the CR2 protein. Binding affinity can be determined by any method known in the art, including for example, surface plasmon resonance, calorimetry titration, ELISA, and flow cytometry. In some embodiments, the molecule has one or more of the following properties of CR2: (1) binding to C3d, (2) binding to iC3b, (3) binding to C3dg, (4) binding to cell-bound fragment(s) of C3b that bind to the two N-terminal SCR domains of CR2.

The molecule described herein is generally capable of inhibiting complement activation of the alternative pathway. The molecule may be a more potent complement inhibitor than the naturally occurring inhibitor protein. For example, in some embodiments, the molecule has a complement inhibitory activity that is about any of 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, or more fold of that of the inhibitor protein. In some embodiments, the molecule has an EC50 of less than about any of 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM. In some embodiments, the molecule has an EC50 of about 5-60 nM, including for example any of 8-50 nM, 8-20 nM, 10-40 nM, and 20-30 nM. In some embodiments, the molecule has complement inhibitory activity that is about any of 50%, 60%, 70%, 80%, 90%, or 100% of that of the inhibitor protein.

Complement inhibition can be evaluated based on any methods known in the art, including for example, in vitro zymosan assays, assays for lysis of erythrocytes, antibody or immune complex activation assays, alternative pathway activation assays, classical pathway activation assays and mannan (lectin pathway) activation assays.

In some embodiments, the molecule is a fusion protein. In some embodiments, the CR2 portion and the inhibitor portion are directly fused to each other. In some embodiments, the CR2 portion and the inhibitor portion are linked by an amino acid linker sequence. Linking sequences can also comprise "natural" linking sequences found between different domains of complement factors. For example, VSVFPLE, the linking sequence between the first two N-terminal short consensus repeat domains of human CR2, can be used. In some embodiments, the linking sequence between the fourth and the fifth N-terminal short consensus repeat domains of human CR2 (EEIF) is used. The order of CR2 portion and inhibitor portion in the fusion protein can vary. For example, in some embodiments, the C-terminus of the CR2 portion is fused (directly or indirectly) to the N-terminus of the inhibitor portion of the molecule. In some

embodiments, the N-terminus of the CR2 portion is fused (directly or indirectly) to the C- terminus of the inhibitor portion of the molecule. In some embodiments, the molecule is a fusion protein. "Fusion protein" used herein refers to two or more peptides, polypeptides, or proteins operably linked to each other. In some embodiments, the targeting portion and inhibitor portion are directly fused to each other. In some embodiments, the targeting portion and inhibitor portion are linked by an amino acid linker sequence. Examples of linker sequences are known in the art, and include, for example, (Gly4Ser), (Gly4Ser)2, (Gly4Ser)3, (Gly3Ser)4, (SerGly4), (SerGly4)2, (SerGly4)3, and (SerGly4)4. Linking sequences can also comprise "natural" linking sequences found between different domains of complement factors. The order of targeting portion and inhibitor portion in the fusion protein can vary. For example, in some embodiments, the C-terminus of the targeting portion is fused (directly or indirectly) to the N-terminus of the inhibitor portion of the targeting construct. In some embodiments, the N-terminus of the targeting portion is fused (directly or indirectly) to the C-terminus of the inhibitor portion of the molecule.

In some embodiments, the molecule comprises a CR2 portion and an inhibitor portion linked via a chemical cross-linker. Linking of the two portions can occur on reactive groups located on the two portions. Reactive groups that can be targeted using a crosslinker include primary amines, sulfhydryls, carbonyls, carbohydrates, and carboxylic acids, or active groups that can be added to proteins. Examples of chemical linkers are well known in the art and include, but are not limited to, bismaleimidohexane, maleimidobenzoyl-N-hydroxysuccinimide ester, NHS-Esters-Maleimide crosslinkers such as SPDP, carbodiimide, glutaraldehyde, MBS, sulfo-MBS, SMPB, sulfo-SMPB, GMBS, sulfo-GMBS, EMCS, sulfo-EMCS, imidoester crosslinkers such as DMA, DMP, DMS, DTBP, EDC and DTME.

In some embodiments, the CR2 portion and the inhibitor portion are non- covalently linked. For example, the two portions may be brought together by two interacting bridging proteins (such as biotin and streptavidin), each linked to a CR2 portion or an inhibitor portion.

In some embodiments, the molecule comprises two or more (same or different) CR2 portions described herein. In some embodiments, the molecule comprises two or more (same or different) inhibitor portions described herein. These two or more CR2 (or inhibitor) portions may be tandemly linked (such as fused) to each other. In some embodiments, the molecule (such a CR2 -inhibitor fusion protein) comprises a CR2 portion and two or more (such as three, four, five, or more) inhibitor portions. In some embodiments, the molecule (such a CR2 -inhibitor fusion protein) comprises an inhibitor portion and two or more (such as three, four, five, or more) CR2 portions. In some embodiments, the molecule (such a CR2 -inhibitor fusion protein) comprises two or more CR2 portions and two or more inhibitor portions.

In some embodiments, there is provided an isolated molecule. In some embodiments, the molecules form dimers or multimers.

The CR2 portion and the inhibitor portion in the molecule can be from the same species (such as human or mouse), or from different species.

In one embodiment, the CR2-inhibitor molecule comprises CR2Crry comprising the amino acid sequence of SEQ ID NO: 16. An exemplary nucleotide sequence encoding CR2Crry is provided in SEQ ID NO: 15. In one embodiment, the CR2-inhibitor molecule comprises a homolog or biologically active fragment of

CR2Crry. Homologs of the CR2Crry include proteins which differ from CR2Crry described herein (or biologically-active fragment thereof) in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition glycosylphosphatidyl inositol). For example, homologue of CR2Crry may have an amino acid sequence that is at least about 70% identical to the amino acid sequence CR2Crry (e.g., SEQ ID NO: 16), for example at least about any of 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% identical to the amino acid sequence of CR2Crry (e.g., SEQ ID NO: 16). Amino acid sequence identity can be determined in various ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNAST AR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment, the CR2-inhibitor molecule comprises human CR2- FH comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment, the CR2-inhibitor molecule comprises a homolog or biologically active fragment of human CR2-FH. Homologs of human CR2-FH include proteins which differ from human CR2- FH described herein (or biologically-active fragment thereof) in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition glycosylphosphatidyl inositol). For example, homologue of human CR2-FH may have an amino acid sequence that is at least about 70% identical to the amino acid sequence human CR2-FH (e.g., SEQ ID NO: 17), for example at least about any of 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% identical to the amino acid sequence of human CR2-FH (e.g., SEQ ID NO: 17). Amino acid sequence identity can be determined in various ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or

MEGALIGNTM (DNAST AR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

In one embodiment, the CR2-inhibitor molecule comprises mouse CR2-

FH comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the CR2-inhibitor molecule comprises a homolog or biologically active fragment of mouse CR2-FH. Homologs of mouse CR2-FH include proteins which differ from mouse CR2- FH described herein (or biologically-active fragment thereof) in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition glycosylphosphatidyl inositol). For example, homologue of mouse CR2-FH may have an amino acid sequence that is at least about 70%) identical to the amino acid sequence mouse CR2-FH (e.g., SEQ ID NO: 18), for example at least about any of 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% identical to the amino acid sequence of mouse CR2-FH (e.g., SEQ ID NO: 18). Amino acid sequence identity can be determined in various ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or

MEGALIGNTM (DNAST AR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

Preparation of CR2-Inhibitor Molecules

The CR2-inhibitor molecules (or the two portions of the CR2 -inhibitor molecules) described herein may be made by chemical synthesis methods, or by linkage of a polynucleotide encoding the CR2 portion and a polynucleotide encoding the inhibitor portion (with or without a linker sequence), and introducing the resulting polynucleotide molecule in a vector for transfecting host cells that are capable of expressing the molecule. Chemical synthesis, especially solid phase synthesis, is preferred for short peptides or those containing unnatural or unusual amino acids such as D-Tyr, Ornithine, and the like. Recombinant procedures are preferred for longer polypeptides. The molecule can be isolated in vitro by protein purification methods. The molecule can also be provided "in situ" by introduction of a gene therapy system to the tissue of interest which then expresses the CR2-inhibitor fusion.

Recombinant DNA techniques for making a CR2-inhibitor fusion protein involves, in simplified form, taking the a CR2-inhibitor encoding polynucleotide, inserting it into an appropriate vector, inserting the vector into an appropriate host cell, and recovering or isolating the fusion protein produced thereby.

Provided herein are polynucleotides that encode a CR2-inhibitor molecule

(i.e., a CR2-inhibitor fusion protein). Such polynucleotide may also be used for delivery and expression of CR2-inhibitor. For example, in some embodiments, there is provided a polynucleotide encoding a fusion protein comprising a CR2 portion comprising a CR2 or a fragment thereof and an inhibitor portion comprising an intact inhibitor molecule or a fragment thereof. In some embodiments, the polynucleotide also comprises a sequence encoding a signal peptide operably linked at the 5' end of the sequence encoding the CR2 -inhibitor fusion protein. In some embodiments, a linker sequence is used for linking the CR2 portion and the inhibitor portion. In some embodiments, the polynucleotide encodes a CR2 -inhibitor fusion protein.

Also provided are expression vectors comprising a polynucleotide described herein for expression of the CR2-inhibitor fusion protein. The expression vector can be used to direct expression of a CR2 -inhibitor fusion protein in vitro or in vivo. The vector may include any element to establish a conventional function of a vector, for example, promoter, terminator, selection marker, and origin of replication. The promoter can be constitutive or regulative, and is selected from, for example, promoters of genes for galactokinase (GALl), uridylyltransferase (GALT), epimerase (GAL 10), phosphoglycerate kinase (PGK), glyceraldehydes-3 -phosphate dehydrogenase (GPD), alcohol dehydrogenase (ADH), and the like.

Many expression vectors are known to those of skill in the art. For example, E. coli may be transformed using pBR322, a plasmid derived from an E. coli species (Mandel et al., J. Mol. Biol., 53 : 154 (1970)). Plasmid pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides easy means for selection. Other vectors include different features such as different promoters, which are often important in expression. For example, plasmids pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pKK233-2 (Clontech, Palo Alto, Calif, USA), and pGEMl (Promega Biotech, Madison, Wis., USA), are all commercially available. Other vectors that can be used in the present invention include, but are not limited to, pET21a (Studier et al., Methods Enzymol., 185: 60-89 (1990)), pRlT5, and pRlT2T (Pharmacia Biotechnology), and pB0475 (Cunningham et al., Science, 243 : 1330-1336 (1989); U.S. Pat. No. 5,580,723). Mammalian expression vectors may contain non-transcribed elements such as an origin of replication, promoter and enhancer, and 5' or 3' nontranslated sequences such as ribosome binding sites, a polyadenylation site, acceptor site and splice donor, and transcriptional termination sequences. Promoters for use in mammalian expression vectors usually are for example viral promoters such as Polyoma, Adenovirus, HTLV, Simian Virus 40 (SV 40), and human cytomegalovirus (CMV). Vectors can also be constructed using standard techniques by combining the relevant traits of the vectors described above. Also provided are host cells (such as isolated cells, transient cell lines, and stable cell lines) for expressing a CR2-inhibitor fusion protein. The host cell may be prokaryotic or eukaryotes. Exemplary prokaryote host cells include E. coli K12 strain 294 (ATCC No. 31446), E. coli B, E. coli XI 776 (ATCC No. 31537), E. coli W3110 (F-, gamma-, prototrophic/ ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species. One suitable prokaryotic host cell is E. coli BL21 (Stratagene), which is deficient in the OmpT and Lon proteases, which may interfere with isolation of intact recombinant proteins, and useful with T7 promoter-driven vectors, such as the pET vectors. Another suitable prokaryote is E. coli W3110 (ATCC No. 27325). When expressed by prokaryotes the peptides typically contain an N-terminal methionine or a formyl methionine and are not glycosylated. In the case of fusion proteins, the N-terminal methionine or formyl methionine resides on the amino terminus of the fusion protein or the signal sequence of the fusion protein. These examples are, of course, intended to be illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for fusion-protein-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacterid., 154(2): 737-742 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC No. 16,045), K. wickeramii (ATCC No. 24,178), K. waltii (ATCC No. 56,500), K. drosophilarum (ATCC No. 36,906; Van den Berg et al., Bio/Technology, 8: 135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP

402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979)); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), and

Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81 : 1470-1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4:475- 479 (1985)). Methyl otropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982). Host cells also include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.

Examples of useful mammalian host cell lines include, but are not limited to, HeLa, Chinese hamster ovary (CHO), COS-7, L cells, C127, 3T3, BHK, CHL-1, NSO, HEK293, WI38, BHK, C127 or MDCK cell lines. Another exemplary mammalian cell line is CHL-1. When CHL-1 is used hygromycin is included as a eukaryotic selection marker. CHL-1 cells are derived from RPMI 7032 melanoma cells, a readily available human cell line. Cells suitable for use in this invention are commercially available from the ATCC.

In some embodiments, the host cell is a non-human host cell. In some embodiment, the host cell is a CHO cell. In some embodiments, the host cell is a 293 cell.

The CR2-inhibitor molecules can be isolated by a variety of methods known in the art. In some embodiments, when the CR2-inhibitor molecule is a fusion protein secreted into the growth media, the molecule can be purified directly from the media. If the fusion protein is not secreted, it is isolated from cell lysates. Cell disruption can be done by any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. The CR2 -inhibitor molecules can be obtained by various methods. These include, but are not limited to, immunoaffinity chromatography, reverse phase chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and HPLC. For example, the CR2-inhibitor molecule can be purified by immunoaffinity chromatography using an antibody that recognizes the CR2 portion or an antibody that recognizes the inhibitor portion, or both. In some embodiments, an antibody recognizing the first two N-terminal SCR domains of CR2 is used for purifying the CR2- inhibitor molecule. In some embodiments, the CR2 -inhibitor molecule is purified by ion change chromatography.

The peptide may or may not be properly folded when expressed as a fusion protein. These factors determine whether the fusion protein must be denatured and refolded, and if so, whether these procedures are employed before or after cleavage. When denaturing and refolding are needed, typically the peptide is treated with a chaotrope, such a guanidine HC1, and is then treated with a redox buffer, containing, for example, reduced and oxidized dithiothreitol or glutathione at the appropriate ratios, pH, and temperature, such that the peptide is refolded to its native structure.

The CR2-inhibitor molecules described herein may also contain a tag

(such as a cleavable tag) for purification. This tag can be fused to the C-terminus or N- terminus of the CR2 portion or the inhibitor portion, and can be used to facilitate protein purification.

In some embodiments, the CR2 -inhibitor molecule could be synthesized de novo in whole or in part, using chemical methods well known in the art. For example, the component amino acid sequences can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid

chromatography followed by chemical linkage to form a desired polypeptide. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing.

The CR2-inhibitor molecules can be assayed for their desired properties using in vitro or in vivo assays. For example, binding of CR2-inhibitor to CR2 ligand can be determined by surface plasmon resonance method. By way of example, kinetic analysis of the interaction of the molecule with C3dg-biotin can be performed using surface plasmon resonance (SPR) measurements made on a BIAcore 3000 instrument (Biacore AB, Uppsala, Sweden). Human C3dg-biotin can be bound to the surface of BIAcore streptavidin sensor chips by injecting C3dg-biotin over the surface of one flow cell of the chip. Binding can be evaluated over a range of CR2-inhibitor molecule concentrations. Association of molecule with the ligand can be monitored for a certain period of time (such as 120 seconds), after which the complex is allowed to dissociate in the presence of buffer only for an additional period of time (such as 120 seconds). Binding of CR2 fusion protein fragments to C3dg-immobilized flow cells can be corrected for binding to control flow cells. Binding data can be fitted to a 1 : 1 Langmuir binding model using BIAevaluation Version 3.1 software (BIAcore) and evaluated for best fit. The kinetic dissociation profiles obtained can be used to calculate on and off rates (ka and kd) and affinity constants (KD) using the BIAevaluation Version 3.1 program. Other assay methods for ligand binding are known in the art and can also be used.

In vitro zymosan complement assay can be used to determine complement inhibitory activity of CR2-inhibitor molecules. Lysis of rabbit erythrocytes by serum in Mg-EGTA is another measure of activity that can be used. Lysis in Mg-EGTA of human or sheep erythrocytes that have had sialic acid removed provides for additional measures of activity.

Targeted molecules in combination with a Thrombolytic agent

In one aspect, the present invention relates to a composition comprising a thrombolytic agent in combination with a targeted molecule described herein. In one embodiment, the composition comprises a sub-therapeutic amount of a thrombolytic agent in combination with a targeted molecule. In one embodiment, the thrombolytic agent is selected from a list comprising but not limited to t-PA, urokinase, anistreplase, ancrod, and brinase.

In certain embodiments, the composition comprises an amount of the thrombolytic agent that is less than the amount necessary when the thrombolytic agent is administered alone. For example, in certain embodiments, the amount or concentration of the thrombolytic agent, when administered in combination with a targeted complement inhibitor described herein, is about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), 90%), or 95% of the amount or concentration of the thrombolytic agent that is efficacious when administered alone.

Uses of CR2 -Inhibitor Molecules and Compositions Thereof

The CR2-inhibitor molecules described herein can function to specifically inhibit in vivo complement activation and inflammatory manifestations that accompany it, such as recruitment and activation of macrophages, neutrophils, platelets, and mast cells, edema, tissue damage, and direct activation of local and endogenous cells.

Compositions comprising these molecules can therefore be used for treatment of diseases or conditions that are mediated by excessive or uncontrolled activation of the

complement system, particularly diseases or conditions mediated by excessive or uncontrolled activation of complement signaling. In some embodiments, there are provided methods of treating diseases involving local inflammation process.

In some embodiments, there is provided a method of treating a disease in which complement pathways implicated in an individual, comprising administering to the individual an effective amount of a composition comprising a CR2-inhibitor molecule comprising: a) a CR2 portion comprising a CR2 or a fragment thereof, and b) an inhibitor portion comprising an inhibitor or a fragment thereof. In some embodiments, there is provided a method of inhibiting complement activation in an individual having a disease in which the alternative complement pathway is implicated, comprising administering to the individual an effective amount of a composition comprising a CR2-inhibitor molecule comprising: a) a CR2 portion comprising a CR2 or a fragment thereof, and b) an inhibitor portion comprising an inhibitor molecule or a fragment thereof. In some embodiments, there is provided a method of inhibiting inflammation in an individual having a disease in which the alternative pathway is implicated, comprising administering to the individual an effective amount of a composition comprising a CR2-inhibitor molecule comprising: a) a CR2 portion comprising a CR2 or a fragment thereof, and b) an inhibitor portion comprising an inhibitor or a fragment thereof.

In one aspect, the present invention provides a method of treating a subject having, or who has had, an ischemic stroke, traumatic brain injury, or spinal cord injury. In certain embodiments, the method comprises administering to the subject one or more of the targeted molecules described herein. In certain embodiments, the method comprises the use of one or more targeted molecules described herein as an adjuvant therapy in combination with one or more standard therapies. For example, in certain embodiments, the one or more targeted molecules are used in combination with rehabilitation therapy. Exemplary types of rehabilitation therapy include, but is not limited to, motor therapy, mobility training, constraint-induced therapy, range-of-motion therapy, electrical and magnetic stimulation, robot-assisted therapy, physical therapy,

occupational therapy, speech therapy, cognitive therapy, visual rehabilitation and the like.

In one embodiment, the method comprises administering one or more targeted molecules as described herein in combination with one or more thrombolytic agents. For example, in one embodiment, the method comprises administering to the subject a composition comprising a targeted molecule and a thrombolytic agent. In one embodiment, the method comprises administering to the subject a first composition comprising a targeted molecule and a second composition comprising a thrombolytic agent.

In certain aspects, the composition is administered to the subject within 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 4 weeks, or more following the onset of stroke or injury. In certain aspects the use of the composition as an adjuvant therapy in combination with one or more other therapies increases the therapeutic window for the treatment of stroke, traumatic brain injury, or spinal cord injury. Administration

The compositions described herein can be administered to an individual via any route, including, but not limited to, intravenous (e.g., by infusion pumps), intraperitoneal, intraocular, intra-arterial, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intra-tracheal, subcutaneous, intrathecal, transdermal, transpleural, topical, inhalational (e.g., as mists of sprays), mucosal (such as via nasal mucosa),

gastrointestinal, intraarticular, intracisternal, intraventricular, rectal (i.e., via suppository), vaginal (i.e., via pessary), intracranial, intraurethral, intrahepatic, and intratumoral. In some embodiments, the compositions are administered systemically (for example by intravenous injection). In some embodiments, the compositions are administered locally (for example by intraarterial or intraocular injection in intracerebral injection). Combination Therapy

In some embodiments, provided pharmaceutical formulations are administered to a subject in combination with one or more other therapeutic agents or modalities, for example, useful in the treatment of one or more diseases, disorders, or conditions treated by the relevant provided pharmaceutical formulation, so the subject is simultaneously exposed to both. In some embodiments, a composition is utilized in a pharmaceutical formulation that is separate from and distinct from the pharmaceutical formulation containing the other therapeutic agent. In some embodiments, a composition is admixed with the composition comprising the other therapeutic agent. In other words, in some embodiments, a composition is produced individually, and the composition is simply mixed with another composition comprising another therapeutic agent.

The particular combination of therapies (substances and/or procedures) to employ in a combination regimen will take into account compatibility of the desired substances and/or procedures and the desired therapeutic effect to be achieved. In some embodiments, provided formulations can be administered concurrently with, prior to, or subsequent to, one or more other therapeutic agents (e.g., desired known

immunosuppressive therapeutics).

It will be appreciated that the therapies employed may achieve a desired effect for the same disorder or they may achieve different effects. In some embodiments, compositions in accordance with the invention are administered with a second therapeutic agent.

As used herein, the terms "in combination with" and "in conjunction with" mean that the provided CR2-inhibitor formulation can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics such as a rehabilitation therapy. In general, each substance will be administered at a dose and/or on a time schedule determined for that agent.

In certain embodiments, the method comprises administering a

composition comprising a combination of a thrombolytic agent and a targeted inhibitor described herein. For example, in one embodiment the method comprises administering a composition comprising t-PA and a targeted inhibitor described herein. In certain embodiments, the method comprises administering one or more compositions. For example, in one embodiment, the method comprises administering a first composition comprising a thrombolytic agent and a second composition comprising a targeted inhibitor described herein. In one embodiment, the method comprises administering a first composition comprising t-PA and a second composition comprising a targeted inhibitor described herein. The different compositions may be administered to the subject in any order and in any suitable interval. For example, in certain

embodiments, the one or more compositions are administered simultaneously or near simultaneously. In certain embodiments, the method comprises a staggered

administration of the one or more compositions, where a first composition is

administered and a second composition administered at some later time point. Any suitable interval of administration which produces the desired therapeutic effect may be used.

In certain embodiments, the method has an additive effect, wherein the overall effect of the administering a combination of therapeutic agents or procedures is approximately equal to the sum of the effects of administering each therapeutic agent or procedure alone. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering a combination of therapeutic agents or procedures is greater than the sum of the effects of administering each therapeutic agent or procedure alone.

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions comprising a CR2- inhibitor molecule and a pharmaceutically acceptable carrier. The pharmaceutical compositions may be suitable for a variety of modes of administration described herein, including for example systemic or localized administration. The pharmaceutical compositions can be in the form of eye drops, injectable solutions, or in a form suitable for inhalation (either through the mouth or the nose) or oral administration. The pharmaceutical compositions described herein can be packaged in single unit dosages or in multidosage forms. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for administration to human. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for intraocular injection. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for topical application. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for intravenous injection. In some

embodiments, the pharmaceutical compositions comprise and a pharmaceutically acceptable carrier suitable for injection into the arteries.

The compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. In some embodiments, the composition is free of pathogen. For injection, the pharmaceutical composition can be in the form of liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the CR2-inhibitor pharmaceutical composition can be in a solid form and redissolved or suspended immediately prior to use. Lyophilized compositions are also included.

For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with

pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

The present invention in some embodiments provides compositions comprising a CR2-inhibitor molecule and a pharmaceutically acceptable carrier suitable for administration to the eye. Such pharmaceutical carriers can be sterile liquids, such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, sodium state, glycerol monostearate, glycerol, propylene, water, and the like. The pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The CR2 -inhibitor molecule and other components of the composition may be encased in polymers or fibrin glues to provide controlled release of the molecule. These

compositions can take the form of solutions, suspensions, emulsions, ointment, gel, or other solid or semisolid compositions, and the like. The compositions typically have a pH in the range of 4.5 to 8.0. The compositions must also be formulated to have osmotic values that are compatible with the aqueous humor of the eye and ophthalmic tissues. Such osmotic values will generally be in the range of from about 200 to about 400 milliosmoles per kilogram of water ("mOsm/kg"), but will preferably be about 300 mOsm/kg.

In some embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for injection intravenously, intraperitoneally, or intracranially. Typically, compositions for injection are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compositions may further comprise additional ingredients, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents, and the like.

Suitable preservatives for use in a solution include polyquaternium-1, benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, benzethonium chloride, and the like. Typically (but not necessarily), such preservatives are employed at a level of from 0.001% to 1.0% by weight.

Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.

Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, and the like, such that the sodium chloride equivalent of the ophthalmic solution is in the range 0.9 plus or minus 0.2%.

Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabi sulfite, sodium thiosulfite, thiourea and the like. Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suitable viscosity-increasing agents include dextran 40, dextran 70, gelatin, glycerin,

hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,

carboxymethylcellulose and the like.

The use of viscosity enhancing agents to provide topical compositions with viscosities greater than the viscosity of simple aqueous solutions may be. Such viscosity building agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose or other agents know to those skilled in the art. Such agents are typically employed at a level of from 0.01% to 2% by weight. In some embodiments, there is provided a pharmaceutical composition for delivery of a nucleotide encoding a CR2-inhibitor molecule. The pharmaceutical composition for gene therapy can be in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle or compound is imbedded.

Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical composition can comprise one or more cells which produce the gene delivery system.

In clinical settings, a gene delivery system for a gene therapeutic can be introduced into a subject by any of a number of methods. For instance, a pharmaceutical composition of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter, See U.S. Pat. No. 5,328,470, or by stereotactic injection, Chen et al. (1994), Proc. Natl. Acad. Sci., USA 91 : 3054-3057. A polynucleotide encoding a CR2 -inhibitor molecule can be delivered in a gene therapy construct by electroporation using techniques described, Dev et al. (1994), Cancer Treat. Rev. 20: 105-115.

Dosing

The optimal effective amount of the compositions can be determined empirically and will depend on the type and severity of the disease, route of

administration, disease progression and health, mass and body area of the individual.

Such determinations are within the skill of one in the art. The effective amount can also be determined based on in vitro complement activation assays. Examples of dosages of CR2 -inhibitor molecules which can be used for methods described herein include, but are not limited to, an effective amount within the dosage range of any of about 0.01 mg/kg to about 300 mg/kg, or within about 0.1 mg/kg to about 40 mg/kg, or with about 1 mg/kg to about 20 mg/kg, or within about 1 mg/kg to about 10 mg/kg. In some embodiments, the amount of CR2FH administered to an individual is about 10 mg to about 500 mg per dose, including for example any of about 10 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 500 mg, about 500 mg to about 1 mg, about 1 mg to about 10 mg, about 10 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, or about 400 mg to about 500 mg per dose.

The CR2-inhibitor compositions may be administered in a single daily dose, or the total daily dose may be administered in divided dosages of two, three, or four times daily. The compositions can also be administered less frequently than daily, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, or once every six months. The compositions may also be administered in a sustained release formulation, such as in an implant which gradually releases the composition for use over a period of time, and which allows for the composition to be administered less frequently, such as once a month, once every 2-6 months, once every year, or even a single administration. The sustained release devices (such as pellets, nanoparticles, microparticles, nanospheres, microspheres, and the like) may be administered by injection or surgical implantation in various locations.

Dosage amounts and frequency will vary according the particular formulation, the dosage form, and individual patient characteristics. Generally speaking, determining the dosage amount and frequency for a particular formulation, dosage form, and individual patient characteristic can be accomplished using conventional dosing studies, coupled with appropriate diagnostics.

Unit Dosages, Articles of Manufacture, and Kits

Also provided are unit dosage forms of CR2- inhibitor molecule compositions, each dosage containing from about 0.01 mg to about 50 mg, including for example any of about 0.1 mg to about 50 mg, about 1 mg to about 50 mg, about 5 mg to about 40 mg, about 10 mg to about 20 mg, or about 15 mg of the CR2-inhibitor molecule. In some embodiments, the unit dosage forms of CR2- inhibitor molecule composition comprise about any of 0.01 mg-0.1 mg, 0.1 mg-0.2 mg, 0.2 mg-0.25 mg, 0.25 mg-0.3 mg, 0.3 mg-0.35 mg, 0.35 mg-0.4 mg, 0.4 mg-0.5 mg, 0.5 mg-1.0 mg, 10 mg-20 mg, 20 mg- 50 mg, 50 mg-80 mg, 80 mg-100 mg, 100 mg-150 mg, 150 mg-200 mg, 200 mg-250 mg, 250 mg-300 mg, 300 mg-400 mg, or 400 mg-500 mg CR2- inhibitor molecule. In some embodiments, the unit dosage form comprises about 0.25 mg CH2- inhibitor molecule. The term "unit dosage form" refers to a physically discrete unit suitable as unitary dosages for an individual, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient. These unit dosage forms can be stored in suitable packaging in single or multiple unit dosages and may also be further sterilized and sealed.

The present invention also provides kits comprising compositions (or unit dosages forms and/or articles of manufacture) described herein and may further comprise instruction(s) on methods of using the composition, such as uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1 : A synergistic effect of complement modulation and rehabilitation on chronic recovery after ischemic stroke

It is demonstrated herein that CR2fH reduces the acute degenerative mechanisms after stroke, thus removing recovery provided a window for rehabilitation therapy to exploit recovery mechanisms. CR2fH treatment reduced acute hemorrhage and improved survival after MCAO

The percentage of animals dying due to hemorrhage (of total acute deaths) was significantly lower in CR2fH treated animals compared to controls (Figure 1 A). CR2fH treatment significantly reduce hemoglobin content in the ipsilateral hemisphere 12 hours (Figure IB) after MCAO compared to vehicle. Nissl stained brain sections showed large hemorrhagic transformation and intracerebral hemorrhage in vehicle controls but not CR2fH treated animals 90 minutes after administration. The combination of CR2fH and rehabilitation significantly enhanced survival at 15 days after MCAO. The survival curve demonstrated that CR2fH treatment 90 minutes after reperfusion alone or in combination with rehab significantly reduced post-stroke mortality up to 15 days after injury. Although rehab alone or delayed administration of CR2fH (6 hours and 24 hours) did not significantly improve survival 15 days after MCAO, combination of rehabilitation and CR2fH treatment at the delayed time points resulted in significant improvement in animal survival further supporting the role of rehab in extending the window of efficacy of CR2fH treatment.

CR2fH treatment (Enriched Environment + skilled handling), significantly reduced infarct exacerbation and scarring after MCAO

Representative T2 -weighted MRI images at days 4 and 14 after MCAO showed large hyper-intense lesions in vehicle and rehab animals compared to CR2fH treated animals with or without rehab (Figure 2A). Quantification of the representative T2-weighted MRI images at days 4 and 14 after MCAO showed significant reduction of hyper-intense lesion volume with CR2fH treatment without rehab. Nissl stained brains showed secondary scarring in the ipsilateral hemisphere 15 days after MCAO that was inhibited by acute CR2fH therapy. Mapping of lesion at different stereotactic coordinates (relative to Bregma) to the Paxinos brain atlas showed the frequency of scarring over serial brain sections for animals within each group (Figure 2C, (c3)). Heat maps map the frequency of overlapping lesions from 8 animals per group. Quantification of lesion volume confirmed histologically that CR2fH reduces secondary scarring 15 days after MCAO with minimal contribution of rehab alone to reduction of scarring. Combination of CR2fH and rehabilitation 15 days after MCAO

The combination of CR2fH and rehabilitation resulted in faster and more pronounced motor recovery 15 days after MCAO. CR2fH administered 90 minutes after ischemia with or without rehab resulted in significant and sustained improvement in neurological deficit over 15 days of recovery compared to vehicle or rehab alone (Figure 3B). Compared to CR2fH, CR2fH with rehab group exhibited a significantly faster recovery during the first week after MCAO. CR2fH also improved recovery of functional deficits when administered 6 hours or 25 hours after MCAO. The comparison of neurological deficits at day 15 after injury showed that CR2fH significantly improved chronic functional recovery compared to vehicle, and once combined with rehab, more pronounced recovery was observed (Figure 3E). These comparisons were made against the vehicle group. A Pair-wise comparison of functional recovery between days 2 and 15 across the different groups showed that despite significant acute improvement achieved with CR2fH alone compared to vehicle, combination of CR2fH with rehab but not rehab alone resulted in more pronounced recovery beyond the acute phase (between days 2 and 15). Among animals treated with rehab, animals co-treated with CR2fH showed significantly more interaction with the enriched environment compared to vehicle treated animals at day 3 but not day 10 after MCAO. During the first week of recovery, CR2fH +/- rehab significantly reduced forelimb asymmetry on corner test compared to vehicle. Mild improvement was seen with rehab alone on day 12 after MCAO compared to vehicle, an effect that was significantly enhanced by combination with CR2fH. Although not significant compared to CR2fH alone, combination treatment has the largest reduction in forelimb asymmetry at any time point. Open field locomotor activity showed a similar pattern as in the first week of recovery with a milder improvement in rehab only group, but a more pronounced improvement in CR2fH+/- rehab groups. No difference was seen in the percent time spent at center controlling for potential difference in anxiety levels between the groups. Using pasta handling task to assess forelimb skilled movement, animals treated with rehab, CR2fH or CR2fH+rehab showed signs of improvement in time to eat compared to vehicle (Figure 3K), but improvement in unilateral handling and number of adjustments was only significant in CR2fH+/- rehab animals compared to vehicle controls.

Improvement in cognitive performance after MCAO with rehab or CR2fH, and more effectively with combination therapy

CR2fH alone or in combination with rehab significantly improved spatial learning (days 9-11 after MCAO) and retention of learned memory (day 15 post-MCAO) or Barnes maze compared to vehicle controls as assessed by the total path length before reaching the target hole (Figure 4A) or the number of error pokes (Figure 4C). Co- treatment with CR2fH and rehab also reduced path length and number of errors significantly compared to rehab alone during learning. At the retention phase, rehab alone showed significant improvement on both measures compared to vehicle whereas

CR2fH+rehab was significantly better than CR2fH alone. Passive avoidance task revealed that animals treated with CR2fH+/- rehab have better memory retention (longer time to enter the shock chamber) compared to vehicle and rehab alone starting 7 days after MCAO. At both 7 and 14 days after injury, CR2fH was significantly better than rehab alone, whereas rehab alone was significantly better than vehicle and combination therapy was significantly superior to all other groups. Principle component analysis of the performance on the different motor and cognitive tasks displayed in Figure 2 and Figure 3 showed that 3 principle components (PC1-PC3) can explain 99.2% of the variance. Individual animal data including shams were plotted against the 3 PCs showing that CR2fH combined with rehab was most efficient at bringing the animals closer to sham compared to either single therapy. PCA analysis performed only on motor or cognitive tasks indicated that the effect of rehab was more pronounced on cognitive rather than motor tasks. The Two principal components explaining around 90% of the variance were plotted (Figure 4F).

CR2fH treatment blocks an inflammatory response after MCAO

CR2fH treatment blocks a robust inflammatory response that would otherwise propagate to the chronic phase after MCAO. Sustained neuroinflammatory response manifested by C3d and IgM deposition 15 days after MCAO was inhibited by single acute administration of CR2fH 90 minutes after MCAO. The quantification of IgM and C3d deposition showed that CR2fH significantly reduce deposition of both markers on the inflamed ipsilateral endothelium. Rehab alone resulted in a mild though significant reduction in IgM and C3d deposition by 15 days after MCAO. GFAP

immunofluorescence staining showed extensive astrogliosis in the peri -infarct area 15 days after MCAO that was significantly inhibited with CR2fH therapy and not rehab alone. Mac2 immunofluorescence staining showed extensive gliosis and proliferation of Ml -polarized (inflammatory type microglia) in the peri-lesional area of vehicle and rehab animals that was significantly inhibited by CR2fH therapy. Quantification of GFAP immunofluorescence and Mac2 immunofluorescence showed that despite that CR2fH more robustly reduces astrocytosis and microgliosis, rehab does have a milder through significant effect on reducing inflammatory phenotype 15 days after stroke.

CR2fH allows maximal effect of rehab therapy

CR2fH removes regenerative mechanisms allowing maximal effect of rehab therapy. The quantified Dcx immunostaining of perilesional hippocampi showed a significant increase in the number of neuroblasts migrating to the ipsilateral hippocampus 15 days after injury with rehab, CR2fH or combination therapy. However, combination of CR2fH and rehab showed more robust increase in Dcx+ cells compared to either single intervention. Immunostaining for markers of regeneration and remodeling including dendritic arborization (MAP2), synaptic density (PSD-95) and axonal growth (GAP-43) of full brain slices showed that combination of CR2fH and rehab resulted in the most pronounced and significant increase in dendritic and axonal growth to the peri-lesional area with subsequent increase in synaptic density. Significant increase in regenerative markers was also seen in CR2fH treated animals (all three marker) and in rehab only animals (MAP2, and PSD95). Co-staining for GAP-43 and GFAP showed that robust astrogliosis inhibits the regrowth of regenerating (GAP -43+) axons into the perilesional area. CR2fH significantly reduced astrogliotic scarring resulting in increased GAP-43 infiltration to peri-lesional brain whereas combination with rehab further increased the levels of GAP-43 in the peri-lesional areas. Rehab only animals showed limited GAP43 increase that was still blocked by astrogliosis. Quantification of GAP-43 density in the peri-lesional area showed that vehicle and rehab animals show a significant reduction in GAP43 density compared to CR2fH+/-rehab surrounding the areas of astrogliosis.

Notable reduction in GAP43 was observed near the areas of astrogliosis in vehicle-treated animals despite comparable neuronal density across the boundaries of the scar

(Neurotrace, cyan). Both CR2fH and rehab alone resulted in significant increase in the levels of BDNF in the ipsilateral hemisphere compared to vehicle; however, combination therapy resulted in a more robust and significant increase compared to either single intervention. Rehab did not influence the levels of TNF-alpha whether alone or in combination with CR2fH treatment. However, CR2fH +/- rehab significantly reduced the levels of TNF-a compared to rehab or vehicle groups.

Immunofluorescence staining of post-mortem human brain sections for IgM and C3d

Immunofluorescence staining of post-mortem human brain sections for

IgM and C3d from patients who died from acute stroke (24-72 hours after onset) showed significant deposition of C3d and IgM specifically in the ipsilateral hemisphere (ischemic penumbra) and not in the contralateral brain from the same patients. The quantification of immunofluorescence showed significantly higher IgM and C3d in the ischemic vs.

control hemisphere.

CR2fH 90 minutes after MCAO

CR2fH administered 90 minutes after MCAO is also protective in female mice and aged male mice (18 months old). CR2fH alone or in combination with rehab significantly reduced neurological deficits in aged mice compared to vehicle; however, only CR2fH with rehab was significantly better compared to rehab only. Survival was significantly improved by CR2fH +/- rehab in aged animals after MCAO. Experiments were terminated at day 10 due to loss of all vehicle controls. CR2fH improved neurological deficits and survival of adult female mice through 15 days after MCAO. Conclusions

CR2fH results in a significant complement inhibitory and antiinflammatory effect after acute stroke leading to reduction in infarct volume and improvement in functional recovery. Rehab in combination with CR2fH treatment resulted in enhanced neuro-regenerative processes (axonal sprouting and neurogenesis) leading to an additive effect on recovery measures, and earlier and more pronounced motor and cognitive recovery. The rehab combination with CR2fH therapy significantly enhanced the window of efficacy of CR2fH for at least 24 hours after injury.

Example 2: The use of CR2-inhibitors in combination with thrombolytic agents

It was examined whether the combination of CR2fH and t-PA provided additional protection and decreased mortality compared to t-PA alone. Neurological deficit scores and forelimb laterality were assessed after t-PA alone (30 minutes after high-dose emboli), CR2fH (30 minutes after high-dose emboli), or CR2fH+t-PA compared to vehicle controls (Figure 9A and Figure 9B). Although single treatment with t-PA or CR2fH significantly reduced neurological deficits and forelimb laterality, combination of CR2fH and t-PA provided a significantly better outcome compared to t- PA alone (Figure 9 A and Figure 9B).

Example 3 : The use of CR2-inhibitors in combination with rehabilitation after traumatic brain injury

Experiments were conducted to examine the effects of complement inhibition and rehabilitation in recovery after traumatic brain injury. Injured mice were assessed on their performance on a spatial learning task (Barnes maze) during chronic recovery from TBI. At 2 months after TBI, animals received 3 doses of vehicle or CR2Crry every other day and then assigned to rehabilitation (enriched environment or regular housing). Animals treated with CR2Crry showed a significantly better learning curve (shorter path length during learning days 83-87) and better retention of learned memory compared to vehicle or rehab alone at 4 weeks after treatment (Figure 10). Repeated measure two-way ANOVA. ***p<0.001. N=7/group. Combination of rehabilitation with CR2Crry also resulted in better cognitive recovery compared to rehabilitation alone. Repeated measure two-way ANOVA. **p<0.01. N=7/group.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.