Neurovascular protection with therapeutic targeting of CD47 / sirpa axis using neutralizing antibodies

Abstract

The present invention provides methods of treating ischemic and traumatic central nervous system injury by administering an antagonist of the interaction between CD47 and SIRPa. Also provided are method of increasing efferocytosis after a traumatic or ischemic CNS injury by administering an antagonist of the interaction between CD47 and SIRPa.

Description

[0001] NEUROVASCULAR PROTECTION WITH THERAPEUTIC TARGETING OF CD471 SIRPA AXIS USING NEUTRALIZING ANTIBODIES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 638,791 filed on April 25, 2024, the contents of which is incorporated by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant number EY035658 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] BACKGROUND

[0007] According to the World Health Organization, ischemic stroke is the second leading cause of death worldwide. It also is a leading cause of adult disability, and many survivors suffer from long-term physical impairment that limits their ability to live independently and enjoy a high quality of life. The economic burden in the United States for ischemic stroke is more than 53 billion dollars per year.1, 2Current therapies focus on restoring blood flow to the brain and reducing neuronal damage. However, these therapies have serious limitations. Thrombolytic medications must be administered by 3 to 4.5 hours after stroke onset and mechanical thrombectomy is only available to eligible stroke patients who present to well-equipped stroke centers within a certain time frame.3’4Thus, there is a strong need for new therapeutics that can be used as solo and / or adjunct therapies to promote brain recovery after ischemic stroke.

[0008] SUMMARY

[0009] The present invention provides method of treating an ischemic or traumatic central nervous system (CNS) injury. In some embodiments, a method of treating atraumatic or ischemic central nervous system (CNS) injury, the method comprising administering an antagonist of the interaction between CD47 and SIRPa is provided. In some embodiments, the antagonist comprises an antibody, a small molecule, an oligonucleotide, a recombinant protein, or an aptamer. In some embodiments the traumatic or ischemic CNS injury comprises stroke, ischemic retinopathies such as diabetic retinopathy, retinal artery or vein occlusion, retinopathy of prematurity, spinal cord injury or traumatic brain injury.

[0010] Another aspect of the present invention provides a method of treating traumatic optic neuropathy, the method comprising administering an antagonist of the interaction between CD47 and SIRPa. In some embodiments, the present invention provides a method of increasing efferocytosis after a traumatic or ischemic central nervous system (CNS) injury, the method comprising administering an antagonist of the interaction between CD47 and SIRPa.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] The present technology can be better understood by reference to the following drawings. The drawings are merely exemplary to illustrate certain features that may be used singularly or in combination with other features and the present technology should not be limited to the embodiments shown.

[0014] Figure 1. Efferocytosis is a process by which myeloid cells (microglia / macrophages) engulf or phagocytose dying or apoptotic cells leading to injury resolution. Myeloid cells identify and engulf apoptotic cells displaying phosphatidylserine (PtdSer) “eat me signal” through the MerTK receptor. Cluster of differentiation 47 (CD47) is a “don’t eat me signal” that is upregulated on apoptotic cells and hinders efferocytosis by binding to its ligand, signal regulatory protein (SIRPa) on myeloid cells. Neutralization of CD47 / SIRPa with antibodies after injury is shown herein to enhance myeloid phagocytosis of apoptotic cells which results in improved outcomes.

[0015] Figure 2. A depiction of the middle cerebral artery occlusion (MCAO) to model ischemic stroke. Scheme of the occlusion of the middle cerebral artery using silicon-coated intraluminal monofilament. A) Simplified scheme of mouse brain and cerebral arteries showing successive sutures and clip to prepare the introduction of silicon-coated monofilament. B) The position of monofilament (in blue color, indicated with arrow) through the circle of Willis is represented. The monofilament is introduced into ICA via ECA to occlude the base of the MCA. ACA, anterior cerebral artery; BA, basilar artery; CCA, common carotid artery; C. Willis, Circle of Willis; ECA, external carotid artery; ICA, internal carotid artery; MCA, middle cerebral artery; PCA, posterior communicating artery; PPA, pterygopalatine artery.

[0016] Figure 3. Experimental protocol for the treatment of MCAO mice.

[0017] Figure 4. CD47 is upregulated in the infarct core after MCAO. A) Coronal section of whole WT mouse brain shows outline of the infarct core exhibiting higher CD47 expression at 24 hr after MCAO. B, C) High magnification insets of the infarct core region confirm higher CD47 expression in the ischemic compared to the contralesional hemisphere. N=4.

[0018] Figure 5. Neutralizing antibodies against SIRPa cross the blood-brain barrier after MCAO. Stroked hemisphere and magnified inset of brain from mouse subjected to 1-hr MCAO and treated at 3 hr with rhodamine labeled anti-SIRPa (8 mg / kg IP). A strong rhodamine signal was detected in the cortical and subcortical areas at 6 hr. There was no signal in the contralesional hemisphere, or mice subjected to sham surgery and treated with labeled antibody or in stroked hemispheres from untreated mice.

[0019] Figure 6. Neutralizing antibodies against the CD47 / SIRPa axis are neuro-protective after MCAO. A, B) Mice subjected to a 1-hr MCAO and treated at 3 hr with anti-CD47 or anti-SIRPa (8 mg / kg IP) show markedly reduced infarct size compared to the anti-IgG control as measured by triphenyltetrazolium chloride (TTC) staining of viable tissue (red / dark) and quantification of infarct area (white). *p<0.05, **p<0.01 vs control. N=6-l l.

[0020] Figure 7. Wire Hang Sensorimotor Assessment. Wire hang shows improved function with anti-SIRPa treatment as compared to anti-IgG. two-way ANOVA *p<0.05 vs anti-IgG, n=3-5 per group.

[0021] Figure 8. Comer Test Sensorimotor Behavioral Assessment. Comer test shows improved function with anti-SIRPa treatment as compared to anti-IgG. two-way ANOVA *p<0.05 vs anti- IgG, n=3-5 per group.

[0022] Figure 9. Novel Object Recognition Test. Anti-SIRPa treatment improves poststroke cognition (time spent with novel object / total exploration time X 100) as compared to anti-IgG. Only mice with exploration time >1 min were included. Student T-test *p<0.05 vs anti-IgG, n=3.

[0023] Figure 10. TNF-a treatment accentuates neuronal CD47 expression after OGD / R insult. CD47 expression increased under normoxic conditions at 24 hr after addition of 20 pg or 50 pg recombinant TNF-a. Expression of CD47 increased after oxygen-glucose deprivation / reoxygenation (OGD / R, 6 hr / 18 hr), a response augmented by TNF-a treatment. Expected MW of CD47 is 35-60 kDa depending on glycosylation level. N=2 per group.

[0024] Figure 11. Temporal Dynamics of Apoptosis, Myeloid Cell Infiltration, and CD47 and TNF-a Expression Following ONC Injury. A) Time course of apoptosis marked by TUNEL (green) labeling on WT retina cross-sections and counterstaining with DAPI (blue) shows progressive accumulation of apoptotic cells (arrows) which peak around day 5 after ONC. B) Myeloid cell response after ONC determined by flatmount immunolabeling at the same timepoints showed activation (enlarged soma) and proliferation of Iba-1 (red) positive myeloid cells (microglia / macrophages). Images are representative of N=3 animals. GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer. C. Time course of TNF-a (membrane bound 26 kDa & trimer 52 kDa forms) levels determined by Western blot on whole retinal lysates shows upregulation of TNF-a starting at day 3 post ONC persisting to 14 days, N=3. D. Western blot on WT retina lysates shows CD47 upregulation at days 4, 5 and 10 after ONC. N=4. E. Immunolabeling of WT retina sections shows strong upregulation of CD47 in the ganglion cell layer (GCL) at day 5 after ONC. F. Immunolabeling of WT optic nerve sections shows upregulation of CD47 at the crush site 7 days after ONC.

[0025] Figure 12. A, B. TNF-a upregulates CD47 in the retinal ganglion cell layer. A-B. Western blot on retinal explants treated with 20, or 50 ng / ml recombinant TNF-a shows significant upregulation of CD47. N=4, *p<0.05 one-way AN OVA. C, D. Immunolabeling of retina explants using NeuN (neuronal marker) shows significant neurodegeneration with 50 ng / ml recombinant TNF-a treatment. N=4, *p<0.05 one-way ANOVA. E-G. Western blotting on retinas from WT mice treated intravitreally with TNF-a neutralizing antibodies after ONC shows decreased TNF- a levels as compared to anti-IgG control. Whole retina lysates showed no difference in CD47 expression between the two groups. N=5, *p<0.05, unpaired t-test, ns=not significant. H, I. Immunolabeling of retinas from WT mice treated with TNF-a neutralizing antibodies after ONC shows downregulation of CD47 in the ganglion cell layer as compared to anti-IgG-treated retinas. N=3-5, *p<0.05, **p<0.0I, ***p<0.001, two-way ANOVA.

[0026] Figure 13. CD47 neutralization facilitates efferocytosis. A-C. Immunolabeling of retina flat mounts from WT mice treated with CD47 neutralizing antibodies shows decreased TUNEL+ apoptotic cells (ACs) and increased efferocytosis index determined by colocalization of ACs with IBA-1+ myeloid cells 5 days post-ONC. The efferocytosis index was measured as the ratio of myeloid cell -associated ACs to free ACs. N=8-9, *p<0.05, unpaired t test D-F. Anti-CD47 showed decreasing trend but not significant reduction of CD45 leukocyte marker and phagocytic marker CD68 ( n=5-7, unpaired t test).

[0027] Figure 14. CD47 neutralization is neuroprotective after ONC. A, B. Retina flatmount immunolabeling at day 14 post ONC using the neuronal marker, NeuN, shows preservation of neurons at the ganglion cell layer with anti-CD47 treatment as compared to anti-IgG control treatment. N=8-9, unpaired t test *p<0.05. C, D. Treatment with anti-SIRPa shows a neuroprotective trend but did not reach statistical significance N=6-8, ns=not significant, unpaired t test. E. Intravitreally injected rhodamine-labeled anti-SIRPa antibodies are retained in the retina up to 7 days after ONC.

[0028] Figure 15. Anti-CD47 treatment rescues retinal thickness and function. A-C. OCT imaging of ONC injured treatment groups B.OCT imaging shows rescue of thickness of the ganglion cell complex (GCC) with anti-CD47 treatment, (n=10-l l per group) unpaired t test, *p<0.05. C.ONC treatment with anti-SIRPa showed a protective trend in GCC thickness but not significant (n=10-12 per group) upaired t test. D. PERG representatives for anti-IgG (control), anti-CD47, and anti-SIRPa treatment groups. E-G. Improved function trend but no significant difference between anti-CD47 control for individual Pl and N2 values. However, there is improved retinal function for anti-CD47 as determined via |P1-N2| (n=9-14 per group) unpaired t test, *p<0.05. H-J. ONC treatment with anti-SIRPa showed improved function trend for P1,N2, and |P 1 -N2| values but not significant (n=10-12 per group).

[0029] Figure 16. Myeloid specific SIRPa deletion improves retinal ganglion cell function after ONC. A-B. hnmunolabeling of retina flatmounts using the neuronal marker, NeuN, shows a neuroprotection trend in the myeloid-specific SIRPa KO mice at day 14 post ONC injury. N=8, unpaired t test, ns=not significant. C-D. OCT imaging shows a trend towards improved ganglion cell complex thickness in the myeloid-specific SIRPa mice vs SIRPaf / flittermate control. N=7, unpaired t test, ns=not significant. Ganglion cell complex = retinal nerve fiber layer (RNFL) + inner plexiform layer (IPL). E-F. PERG representatives and analysis show improved retinal function in SIRPa mice vs SIRPaf / flittermate controls at day 14 after ONC determined as |P1- N2|. N=9-14, unpaired t test, *p<0.05.

[0030] DETAILED DESCRIPTION

[0031] The present invention provides method of treating an ischemic or traumatic central nervous system (CNS) injury. The inventors found a strong protective effect using neutralizing antibodies for the cluster of differentiation 47 (CD47) and its binding partner, signal regulatory protein (SIRPa), in two different preclinical models of CNS diseases- stroke and traumatic optic neuropathy.

[0032] CD47 is an integrin-associated protein ubiquitously expressed on neurons and all other cell types in the human body. CD47 binds to ligand SIRPa, a regulatory membrane glycoprotein expressed on the surface of microglia and macrophages (collectively known as myeloid cells). CD47-SIRP signaling works as an immune checkpoint by which myeloid cells can recognize cells displaying CD47 as “self’. Structurally, the five-fold transmembrane protein CD47 contains a larger extracellular N-terminal domain than the short intracytoplasmic C-terminal tail. The extracellular IGV-like domain on CD47 binds to SIRPa on myeloid cells, including macrophages, to initiate phosphorylation of two tyrosine residues of immune receptor tyrosine inhibitory motifs (ITIMs) in the intracellular domain of SIRPa. Phosphorylation of ITIMs re-recruits and activates the phosphatase Src homology 2-containing protein tyrosine phosphatase- 1 (SHP-1) and SHP-2, impeding phagocytosis potentially via deactivation of non-muscle myosin-IIA.

[0033] Blocking CD47 / SIRPa binding results in a failure in the recruitment and activation of SHP1 / 2, thereby promoting phagocytosis. During this process, cross-priming of antigen- presenting cells (APCs) is also initiated, leading to activation of the adaptive immune system. In vitro and in vivo studies showed that CD47 / SIRPa blocking antibody significantly enhanced macrophage phagocytosis, dendritic cell (DC) cross-presentation, and neutrophil-mediated killing. Efferocytosis, the phagocytosis of apoptotic cells, is an important process for inflammatory response resolution and tissue repair after injury. Apoptotic cells express pro- efferocytotic “eat me” signals, such as phosphatidylserine (PtdSer). These signals encourage phagocytosis when they bind to ligands, such as MerTK, on myeloid cells. Since efferocytosis relies on these “eat me” signals to clear apoptotic cells, blockade of anti-efferocytotic “don’t eat me” signaling, such as CD47-SIRP axis, has the potential to increase the efficacy of efferocytosis after injury. This rapid clearance of dying cells reduces the deleterious effect of inflammation caused by lingering apoptotic cells.

[0034] Efferocytosis plays a vital role in health and disease. The protective role of efferocytosis in CNS pathological conditions has been elucidated in recent studies. Incomplete clearance of apoptotic cells in CNS injuries leads to a feed-forward cycle of inflammation and furthers cell death. Recent studies have demonstrated the beneficial effect of CD47 or its ligand SIRPa neutralization in cancer and atherosclerosis models. Anti-CD47 and anti-SIRPa antibodies are now utilized in clinical trials and preclinical trials as a treatment for these conditions. However, the role of CD47 / SIRPa in efferocytosis has not been studied in ischemic stroke or traumatic optic neuropathy. Similarly, CD47 and SIRPa antibodies are being used in clinical trials for certain malignancies but have never been used to treat ischemic stroke or traumatic optic neuropathy. Data described herein demonstrates that CD47, SIRPa and or TNFa is upregulated following optic nerve injury. In some embodiments, CD47 is upregulated as soon as 2, 4, 6, 8, 10, 12, 18, 24 or 48 hours after injury or may be upregulated 4, 5, 6, 7, 14, 21 or 28 or more days, and any number in between after optic nerve injury. CD47 may be upregulated in any effected cell, including, but not limited to ganglion cells, retinal microglia, myeloid cells, monocytes, dendritic cells, neutrophils, other glia cells and neurons.

[0035] Methods:

[0036] In one aspect, the present disclosure provides a method of treating traumatic or ischemic central nervous system (CNS) injury. In some embodiments, the method comprises administering an antagonist of the interaction between CD47 and SIRPa. As used herein, an antagonist is a structure or agent that interferes with or stops the action of another. Antagonizing may be used interchangeably with antagonist. A method of antagonizing may include interfering with, preventing, blocking or reducing the physiological action of another. An antagonist may be reversible or irreversible. A method of antagonizing the interaction between CD47 and SIRPa is described herein, wherein binding between CD47 and SIRPa is decreased or prevented. An antagonist of the CD47 SIRPa interaction may directly or indirectly block the binding of CD47 and SIRPa. An antagonist of the CD47 SIRPa interaction may inhibit or reduce the expression of CD47 or SIRPa at the transcriptional, translational or post-translational levels. An antagonist of the CD47 and SIRPa may also inhibit or reduce SIRPa downstream signaling, such that phagocytic activity is maintained or increased, or inflammatory cytokine production is decreased, for example TNFa.

[0037] Examples of CD47 and SIRPa antagonists include but are not limited to antibodies, small molecules, oligonucleotides, recombinant proteins, and aptamers that may bind to one of CD47 or SIRPa and inhibit the interaction between the two proteins. In some embodiments the antagonist comprises an anit-CD47 or an anti-SIRPa antibody. An anti-CD47 or an anti-SIRPa antibody may be a neutralizing or inhibitory antibody. By way of example and not limitation, the antagonist antibody may comprise TTI-621, TTI-622, Hu5F9-G4, ALX148 or CC-95251. In some embodiments, the antagonist may comprise a small molecule. By way of example and not limitation, the antagonist may comprise NCGC00138783, PEP-20 or D4-2.

[0038] Methods provided herein may be used to treat a traumatic or ischemic CNS injury. A traumatic injury may be the result of an external force, for example a blunt or penetrating force which causes injury. An ischemic injury occurs when the blood supply to an area or tissue is cut off or reduced. Without limitation examples of traumatic or ischemic CNS injuries include, but are not limited to stroke, ischemic retinopathy such as diabetic retinopathy, retinal artery or vein occlusion, retinopathy of prematurity, spinal cord injury or traumatic brain injury. In some embodiments, the injury is an ischemic stroke.

[0039] Ischemic stroke occurs when a blood clot, or thrombus, blocks or plugs an artery leading to the brain. Lack of blood supply, and thus oxygen to the brain causes the brain cells and tissue to die. Ischemic strokes include thrombotic strokes, embolic strokes and transient ischemic strokes. Ischemic stroke is different from hemorrhagic stroke. One difference between the two types of stroke is the underlying cause of the brain damage. In ischemic stroke, the damage is caused by a lack of blood supply, while in hemorrhagic stroke, it is caused by bleeding into the brain tissue. Treatment is also different between the two types of stoke. Treatment for an ischemic stroke should begin as soon as possible, ideally within 3 hrs of the stroke starting. Treatment for ischemic stroke includes thrombolytic drugs to dissolve or break up the clot. Treatments may also include blood thinners. In some embodiments the present invention provides methods of treating ischemic stroke comprising administering an antagonist of the interaction between CD47 and SIRPa. In some embodiments the antagonist is administered within 3 hours of the ischemic stroke, or other CNS injury. In some embodiments, the antagonist is administered within 6 hours, 12 hours, 24 hours, 48 hours, 72 hours or 96 hours of the CNS injury.

[0040] Another aspect of the present invention provides a method for treating traumatic optic neuropathy. In some embodiments, the method comprises administering an antagonist of the interaction between CD47 and SIRPa. In some embodiments, the method improves visual acuity, color vision, visual field deficit, axonal regeneration or neuroprotection increases. In some embodiments, the method decreases optic atrophy. In some embodiments the antagonist is administered within 1 week of atraumatic optical injury. In some embodiments, the antagonist is administered within 5 days, 4 days, 3 days, 2 days or 1 day of the traumatic injury.

[0041] Another aspect of the present invention provides a method of increasing efferocytosis after a traumatic or ischemic central nervous system (CNS) injury. In some embodiments, the method comprises administering an antagonist of the interaction between CD47 and SIRPa. Without limitation the traumatic or ischemic CNS injury may comprise a stroke, ischemic retinopathy, spinal cord injury, traumatic brain injury or traumatic optical injury.

[0042] An antagonist of the CD47 SIRPa interaction may be administered as an adjuvant therapy, along with other standard of care for a traumatic or ischemic CNS injury. For example, an antagonist may be administered before, during or after a thrombolytic drug or blood thinner. A CD47 SIRPa antagonist may be administered within hours or days after the first signs of a stroke or traumatic injury. In some embodiments, the antagonist may be administered one or more times.

[0043] In some embodiments, the antagonist is administered within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 32 hours, or 48 hours, 3 days, 4 days, or 5 days of a CNS injury.

[0044] As used herein, the term "administering" an agent, such as a CD47 SIRPa antagonist is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the subject, including delivery by either the parenteral or oral route, intravitreal, perioperative, epidural, intracerebral, intracerebroventricular, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route. Administration may be systemic or localized.

[0045] As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. The term "treat" further includes the reduction in one or more symptom associated with traumatic or ischemic CNS injury, including stroke, ischemic retinopathy, spinal cord injury, traumatic brain injury or traumatic optical injury.

[0046] In some embodiments treatment with an CD47 SIRPa antagonist reduces infarction area, swelling, or recovery time. In some embodiments treatment increases cognitive recovery and / or motor function recovery. In some embodiments, treatment improves visual acuity, color vision or axonal regeneration or decreases optic atrophy. In some embodiments, treatment is neuroprotective. Neuroprotective treatments preserve neuronal structures and / or function and reduce the rate or amount of neuronal loss. Treatments provided herein may be combined with other known neuroprotective therapies.

[0047] The present invention also provides a composition comprising an antagonist of the CD47 SIRPa interaction for use in preparation of a medicament for treatment of ischemic CNS injury or traumatic CNS injury. In some embodiments, the ischemic CNS injury comprises an ischemic stroke. In some embodiments the traumatic CNS injury comprises optic neuropathy.

[0048] Additional definitions

[0049] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps.

[0050] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter.

[0051] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus < 10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0052] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0053] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0054] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g. , “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0055] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0056] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0057] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0058] EXAMPLES

[0059] Central nervous system (CNS) injury from ischemia or trauma can affect the brain and retina leading to morbidity and disability. During an acute ischemic stroke, the transient loss of blood flow results in an inadequate supply of oxygen and glucose to the brain. Traumatic optic neuropathy (TON) is an injury to the optic nerve and is often the result of direct or indirect trauma and leads to vision loss, which can be permanent. CNS injury in both conditions is characterized by inflammation and neuronal degeneration. These conditions have different etiologies but are similarly characterized by inflammation, neurodegeneration, and myeloid cell activation / infiltration. Therefore, both injuries offer unique insight into the myeloid cell response in the central nervous system (CNS) after ischemia and trauma.

[0060] Efferocytosis is a reparative process by which myeloid cells remove apoptotic cells (AC) by phagocytosis after tissue injury. Efferocytosis is a highly evolutionarily conserved process that maintains tissue homeostasis.1Myeloid cells, microglia, and macrophages are known as professional phagocytes and are integral in the clearance of AC. Under healthy conditions, “eat me” signals such as phosphatidylserine (PtdSer) displayed on AC encourage engulfinent of the dying cell when it binds to MER tyrosine kinase (MerTK) on myeloid cells. Cluster of differentiation 47 (CD47) is an integrin-associated protein that is commonly known as the “don’t eat me” signal. It binds to signal regulatory protein (SIRP) on the surface of myeloid cells. This helps professional phagocytes recognize cells as self and spares them from phagocytosis (Fig. I).2Upregulation of (CD47) has been implicated in dysfunctional efferocytosis in diseases such as cardiovascular disease and cancer.3,4Currently, CD47 antibodies are being used clinically and preclinically as a therapeutic to neutralize CD47 in cancer and atherosclerosis. Tumor necrosis factor- (TNF-) has been implicated in CD47 upregulation in these conditions43. Yet, the lack of understanding of the mechanisms involved in clearing apoptotic cells after CNS injury is a major barrier to developing novel clinically effective treatments. Thus, it is important to establish and characterize the role of the CD47 and SIRP axis in retinal neurodegeneration after TON and ischemic stroke to further elucidate immune cell interaction in the CNS after injury.

[0061] The inventors have found that mouse retinas subjected to TON injury show upregulation of the cluster of differentiation 47 (CD47) protein, a “don’t eat me” signal which inhibits phagocytosis when it binds to its ligand, signal regulatory protein a (SIRPa) on myeloid cells. They have also found that tumor necrosis factor- (TNF-) increases in concurrence with CD47 in these retinas (unpublished). They obtained similar results in a mouse stroke model. This leads them to hypothesize that CD47 upregulation in CNS injury is driven by TNF- and disrupting CD47-SIRPa interaction in CNS injury may enhance efferocytosis, thus providing neuronal protection.

[0062] Described herein, the inventors use optic nerve crush (ONC), a TON model (Figs. 12 and 13), and middle cerebral artery occlusion (MCAO) an ischemic stroke model (Figs. 2 and 3) to investigate CD47 regulation as well as the impact of modulation on CD47 / SIRP axis in CNS efferocytosis.

[0063] Determining the role of CD47 and SIRP in TON and ischemic stroke is novel and therefore this proposal carries significant potential to move the field forward by proposing new therapeutics and elucidating mechanisms of CD47 increase in CNS injuries.

[0064] Example 1: Middle Cerebral Artery Occlusion model of ischemic stroke and the role of CD47 / SIRPa axis in CNS injury

[0065] Initial studies indicate that CD47 global gene deletion is protective against focal cerebral ischemia in mice. These studies have focused on the endothelial cytotoxic and anti-angiogenic effects of CD47 through binding to the ligand, thrombospondin 1 (TSP-1), thus emphasizing that CD47 may play diverse pathological roles in stroke. However, no studies addressed the role of myeloid SIRPa in stroke to date. It has recently been found that blockade of CD47 with the use of an antibody accelerates hematoma clearance post intracerebral hemorrhage, and depletion of myeloid cells increased brain swelling and neurodegeneration but the pathology and treatment of intracerebral hemorrhage is completely different from ischemic stroke.22This highlights the importance of efferocytosis in CNS injury and shows the additional benefit of blocking CD47 in intracerebral hematomas which is difficult to diagnose from ischemic stroke without brain scans.23Therefore, anti-CD47 can be administered in ambulance prior to differential diagnosis.

[0066] Results:

[0067] The potential contribution of the CD47 / SIRP axis to impaired efferocytosis and neuronal cell death has not been studied in ischemic stroke to our knowledge. CD47 is known to inhibit phagocytosis when it binds to SIRP on the surface of professional phagocytes including microglia and macrophages.

[0068] Our results show upregulation of CD47 in the stroked hemisphere (Fig. 4). One of our other key observations is that the anti-SIRPa neutralizing antibody, administered systemically (8 mg / kg, IP) at 3 hr after MCAO, crosses the blood brain barrier (Fig. 5) and provides robust neuroprotection with reduction in infarct area compared to anti-IgG treated control animals (Fig. 6). These findings implicate the CD47 / SIRP axis as a contributor to infarct size after ischemic stroke.

[0069] The hypothesis that TNF-a upregulates CD47 in neurons is supported by western blot, using the differentiated R28 neuronal cell line (Fig. 10) . In this preparation, addition of TNF-a (20 and 50 pg) for 24 hr appeared to increase CD47 expression under normoxic conditions (Fig. 10, 3 left lanes). Interestingly, expression of CD47 increased after oxygen-glucose deprivation / reoxygenation for 6hr / 18hr (OGD / R; an in vitro model of neuronal hypoxia), and expression further increased after the addition of recombinant TNF-a (Fig. 10, 3 right lanes). This finding implies that TNF-a can induce CD47 expression in R28 neuronal cells.

[0070] Materials and Methods:

[0071] MCAO is performed unilaterally under anesthesia. Surgical filament is inserted through the external carotid artery and threaded through the internal carotid artery until it reaches the middle cerebral artery. Filament used is Doccol Silicone rubber-coated monofilament for MCAO model. Filament size 6-0, diameter 0.09-0.11 mm, length 20mm; diameter with coating 0.21 + / - 0.02 mm; coating length 4-5 mm, suitable for MCAO with reperfusion. The filament will occlude the middle cerebral artery origin for one hour, then removed.32Sham MCAO procedure (insertion and immediate removal of filament) is used for control. Intraperitoneal Anti-SIRPa or anti-IgG dose of 200 pg (BioXCell) will be administered at 3 hours, post MCAO, according to the published literature.30We have chosen 1-hour occlusion model based on our preliminary experiments (Fig. 2 & 3) to achieve robust ischemia reperfusion injury and behavioral deficit.

[0072] Myeloid-specific SIRP KO mice: Myeloid-specific SIRPa KO mice under a tamoxifen-inducible Cx3crl ere will be used to investigate the role of SIRPa on myeloid cells in mouse injury models of TON and ischemic stroke.

[0073] • These mice will be male and female mice aged 8 weeks. These mice were received via material transfer from Hisashi Umemori, Ph.D., Harvard Medical School. Tamoxifen administration will be conducted by intraperitoneal (IP) injection of 75 mg / kg twice (one day apart) using a 20 mg / mL stock of tamoxifen in com oil.25,26Experiments will be conducted 6 days after the last dose of tamoxifen when myeloid Cre activity is high, and tamoxifen is eliminated from the circulation since tamoxifen has been found to be neuroprotective.27Cre negative littermates with the floxed gene will be used as controls in each study. Cx3crlCreERT2 controls also will be examined and compared to floxed controls to account for potential abnormal effects of Cre transgene expression. Tamoxifen will be administered to the KO and control groups to account for any off-target effects associated with the treatment.

[0074] Primary mouse embryonic neurons: Will be isolated and collected from wild-type mouse embryos (El 7) and cultured on polyethyleneimine coated plates in neurobasal NBM+B27 (Neurobasal medium, lx B27 supplement, 500 mM glutamine, and 10 mg / ml gentamycin).33Bone marrow derived macrophages (BDMD): will be isolated from the femurs ofWT mice and differentiated via differentiation medium (DMEM high glucose containing 20% FBS, 20% L929 cells conditioned media, and 1% P / S).34

[0075] Efferocytosis Index: Efferocytosis will be tested with immunolabeling for Ibal (myeloid cell marker), and TUNEL kit to label apoptotic cells. Efferocytosis parameters will be counted in high- power z-stack images per section digitized from the ischemic peri-infarct penumbra area for stroke and from flat mounts of retinal ganglion cell layer. Efferocytosis index (% of dead / dying cells engulfed by microglia / M<b) will be calculated using the following equation: ([number of Ibal+TUNEL+ cells / number of TUNEL+ cells] x 100%). The Efferocytosis index will be used for ONC and MCAO experiments.

[0076] Flow cytometry: Flow cytometry will be used to determine immune cell phenotypes in ONC and MCAO experiments.35Mice will receive ketamine (80 mg / kg) and xylazine (8 mg / kg) and will undergo transcardial perfusion to remove intravenous immune and blood cells35. Injured retinas and brain hemispheres will be compared against sham retinas and brains.

[0077] Stroke behavioral testing: A battery of behavioral tests will be used to assess brain function and motor coordination before and after stroke. These include rotarod, wire hang, comer test, beam walk, modified Bederson, and grid walk tests and will be carried out as recommended by the Stroke Preclinical Assessment Network (SPAN). 36 o Rotarod is a test in which mice are placed on a rod that incrementally increases speed up to 40 RPM in 5 minutes. Mice will be trained on rotarod 5 days prior to stroke and results post-stroke will be compared to individual results prior to stroke. o The comer test (Fig. 8) will be used to identify unilateral sensory and motor deficits. Mice will be placed in a 30° comer and directional turns out of the comer will be tabulated. The comer test will be administered prior to stroke to establish no preexisting directional bias. The beam walk test is a balance test in which mice walk from one side of the beam to the other, coordination is scored from 0 (no deficit) to 6 (falling from the beam <20 seconds). o The modified Bederson is a test in which mice are placed in an open field environment and behavior is scored from 0 (no deficit) to 5 (no spontaneous movement indicative of severe neurological deficit). Tests will be administered starting at 24 hours post-stroke and tested once weekly until 30th day post stroke. o For wire hang test (Fig. 7), mice are turned upside down and the latency to when the mouse falls is recorded. Tests will be administered starting at 24 hours post-stroke and tested once weekly until 30th day post stroke. o The grid walk test scores coordination. Mice will be placed on a grid for 5 minutes and foot faults (slips through the grid) will be tabulated. Tests will be administered starting at 24 hours post-stroke and tested once weekly until 30th day post stroke. o To test cognitive function, mice will be tested for novel object recognition (NOR) (Fig. 9). At 30 days post ischemic stroke, mice will receive a training day with exposure to two of the same type of object. The following day, one of the objects will be replaced with a new but similar object. This will challenge the mouse’s recognition memory and is an effective way to evaluate cognition.

[0078] Oxygen Glucose Deprivation (OGD): To mimic the ischemic stroke in vitro, we will subject primary cortical neurons to OGD. For glucose deprivation, we will use glucose free DMEM. For oxygen deprivation, cells will be placed in a humidified 37 °C chamber with <1% 02, 94% N2, and 5% CO2 for desired duration, followed by reoxygenation in a humidified 37 °C incubator with 95% air, 5% CO2 at 37 °C.

[0079] Efferocytosis In Vitro Assay: To mimic efferocytosis in vitro, we will serum starve primary cortical neurons to induce apoptosis, then coculture these cells with differentiated BMDM. We will treat with anti-CD47, anti-SIRP, or anti-IgG for control. Western blot, TUNEL staining (apoptotic cell marker) and IHC staining using NeuN (neuronal marker) and Iba-1 (myeloid marker) will be performed after 24 hours of incubation. NeuN staining will establish degree of neuroprotection with antibody treatment. TUNEL and Iba-1 colocalization will determine the efficiency of efferocytosis with treatment.

[0080] References for Example 1:

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[0104] 26. Valny M, Honsa P, Kirdajova D, Kamenik Z, Anderova M. Tamoxifen in the mouse brain: Implications for fate-mapping studies using the tamoxifen-inducible cre-loxP system. Frontiers in Cellular Neuroscience . 2016;10:243. doi: 10.3389 / fhcel.2016.00243.

[0105] 27. Wakade C, Khan MM, De Sevilla LM, Zhang Q, Mahesh VB, Brann DW. Tamoxifen neuroprotection in cerebral ischemia involves attenuation of kinase activation and superoxide production and potentiation of mitochondrial superoxide dismutase. Endocrinology (Philadelphia). 2008; 149(l):367-379. doi: 10.1210 / en.2007-0899.

[0106] 28. Fouda AY, Eldahshan W, Xu Z, et al. Preclinical investigation of pegylated arginase 1 as a treatment for retina and brain injury. Experimental neurology. 2022;348: 113923. doi: 10. 1016 / j .expneurol .2021. 113923.

[0107] 29. Xu Z, Fouda AY, Lemtalsi T, et al. Retinal neuroprotection from optic nerve trauma by deletion of arginase 2. Frontiers in Neuroscience. 2018;12:970. doi: 10.3389 / fnins.2O18.00970.

[0108] 30. Liu J, Xavy S, Mihardja S, et al. Targeting macrophage checkpoint inhibitor SIRPa for anticancer therapy. JCI insight. 2020;5(12). doi: 10.1172 / j ci. insight.134728.

[0109] 31. Tang Z, Zhang S, Lee C, et al. An optic nerve crush injury murine model to study retinal ganglion cell survival. Journal of Visualized Experiments . 2011(50). doi: 10.3791 / 2685.

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[0111] 33. Fouda AY, Pillai B, Dhandapani KM, Ergul A, Fagan SC. Role of interleukin- 10 in the neuroprotective effect of the angiotensin type 2 receptor agonist, compound 21, after ischemia / reperfusion injury. European journal of pharmacology. 2017;799: 128-134. doi: 10.1016 / j.ejphar.2017.02.016.

[0112] 34. Fouda AY, Xu Z, Shosha E, et al. Arginase 1 promotes retinal neurovascular protection from ischemia through suppression of macrophage inflammatory responses. Cell Death & Disease. 2018;9(10): 1001-15. doi: 10. 1038 / s41419-018-1051-6.

[0113] 35. Kipp M, Kiessling MC, Hochstrasser T, Roggenkamp C, Schmitz C. Design-based stereology for evaluation of histological parameters. J Mol Neurosci. 2017;61(3):325-342. doi: 10. 1007 / S12031-016-0858-9.

[0114] 36. Lyden PD, Bosetti F, Diniz MA, et al. The stroke preclinical assessment network: Rationale, design, feasibility, and stage 1 results. Stroke (1970). 2022;53(5): 1802-1812. doi: 10. 1161 / STROKE AHA.121.038047.

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[0123] Example 2: Disrupting the CD47 / SIRPa Axis Protects Against Traumatic Optic Neuropathy

[0124] Traumatic optic neuropathy (TON) is a serious injury to the optic nerve that can lead to vision impairment or blindness. Current treatment options include corticosteroids or surgical decompression. However, there is no consensus for the treatment of TON due to a lack of evidence of improvement with current options. Cluster of differentiation 47 (CD47) is a transmembrane protein that can be upregulated on apoptotic cells. It prevents their phagocytic clearance or efferocytosis by communicating a “don’t eat me” signal when it binds to signal regulatory protein a (SIRP a) on myeloid cells. We found that CD47 is upregulated in the retina after optic nerve crush (ONC) injury along with the pro-inflammatory cytokine tumor necrosis factor- a (TNF- a). Antibody neutralization of TNF- a after ONC resulted in blunted CD47 levels whereas treating ex-vivo retinal explants with recombinant TNF- a resulted in increased levels of CD47, suggesting that TNF- a plays a stimulatory role in CD47 expression. To investigate the therapeutic potential of blocking the CD47 / SIRP a axis after ONC, we intravitreally treated WT mice with anti-CD47, anti-SIRPa, or anti-IgG (control) post-ONC injury. Anti-CD47 and anti-SIRP a treatments resulted in improved efferocytosis, attenuated neurodegeneration, improved retinal thickness, and enhanced retinal function to varying degrees as compared to anti-IgG control treatment post-ONC. Myeloid-specific SIRP a deletion showed a neuroprotective trend and resulted in significantly improved retinal ganglion cell function after ONC. Collectively, our data suggest that the CD47- SIRP a axis is a promising therapeutic target to improve retinal structural and functional outcomes after ONC injury.

[0125] Introduction

[0126] Traumatic optic neuropathy is a condition where the optic nerve is damaged due to direct or indirect trauma. The optic nerve is a bundle of nerve fibers that transmits visual information from the eye to the brain. TON can occur due to a variety of causes, including blunt or penetrating trauma, compression, or stretching of the optic nerve. The symptoms of TON can include sudden vision loss, pain around the eye, and changes in color perception. TON is often irreversible, and vision loss is permanent.7TON is characterized by inflammation, immune response and neuronal degeneration following injury. The lack of understanding of the mechanisms involved in tissue injury and repair after TON is a major barrier to developing novel clinically effective treatments.

[0127] Efferocytosis is the phagocytosis of apoptotic cells (AC). It is a highly evolutionarily conserved process that maintains tissue homeostasis.1Myeloid cells, including microglia and macrophages, are known as professional phagocytes and are integral in the clearance of AC. Under healthy conditions, “eat me” signals such as phosphatidylserine (PtdSer) displayed on AC encourage engulfment of the dying cell by myeloid cells.10Efferocytosis is vital for inflammatory response resolution and tissue repair after injury. Since efferocytosis relies on these “eat me” signals to clear apoptotic cells, blockade of anti-efferocytotic “don’t eat me” signaling on apoptotic cells, such as Cluster of differentiation 47 (CD47) - signal regulatory protein (SIRP ) axis, has the potential to increase the efficacy of efferocytosis after injury. The rapid clearance of dying cells reduces the deleterious effect of inflammation caused by lingering apoptotic cells.

[0128] CD47 is an integrin-associated protein ubiquitously expressed on neurons and other cell types in the human body. It is a commonly known “don’t eat me” signal.2CD47 binds to the ligand SIRPa, a regulatory membrane glycoprotein expressed on the surface of microglia and macrophages. CD47-SIRP a signaling works as an immune checkpoint by which myeloid cells can recognize cells displaying CD47 as “self’.8When CD47 binds to SIRP a, the tyrosine residues of SIRP a are phosphorylated, activating Src homology 2-containing protein tyrosine phosphatase- 1 (SHP-1) and SHP-2, impeding phagocytosis potentially via deactivation of nonmuscle myosin-IIA.9Upregulation of CD47 has been implicated in dysfunctional efferocytosis in cardiovascular diseases and cancer.3,4Tumor necrosis factor- a (TNF- a) has been implicated in CD47 upregulation in these conditions.43Yet regulation of CD47 in retinal injury is still ill- defined. Recent studies explored the role of CD47 in mouse models of retinitis pigmentosa and autoimmune uveitis.23,33Interestingly, CD47 augmentation protects cone photoreceptors from genetic retinal degeneration whereas its deficiency ameliorates ocular autoimmune inflammation, emphasizing that CD47 may play diverse and disease-specific roles in ocular conditions that we cannot fully untangle at this time. However, the contribution of the CD47-SIRPa axis to injury progression in TON is unknown.

[0129] In this study, we demonstrate that CD47 levels are increased in the inner retina following traumatic optic neuropathy (TON), which inhibits efferocytosis and the repair of retinal tissue. By disrupting the CD47-SIRPa axis through the use of neutralizing antibodies or by specifically deleting SIRPa in myeloid cells, we are able to protect against retinal neurodegeneration and improve retinal function after TON. These finding provides proof of concept that targeting the CD47-SIRPa pathway could be a promising therapeutic approach for treating TON.

[0130] Materials and Methods:

[0131] Animals

[0132] The Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences approved all animal experiments. Mice were housed at 25 °C with light / dark cycle and fed ad libitum maintenance chow. Wild-type (WT) C57BU / 6J male mice were purchased from Jackson Uaboratory (Bar Harbor, ME, USA).

[0133] Generation of myeloid-specific SIRPa KO mice

[0134] SIRPa / 7 / 7on a C57BE / 6J background (obtained from Dr. Hisashi Umemori at Harvard Medical School) were used to generate myeloid-specific SIRPa KO mice (M-SIRPa7)(PMID: 24036914). SIRPa7777mice were bred with mice expressing tamoxifen-inducible Cre recombinase under the direction of the Cx3crl promoter [Jackson Uaboratory Stock No: 020940 (Cx3crlCreERT2)60as described above to generate M<b and microglia cell-specific SIRPa knockout (Cx3crlCreERT2; mice were used as controls. We have characterized these mice and confirmed that they display normal retina morphology using histology. To account for any off-target, both SIRPa KO and SIRPa / 7 / ?micc were treated with Tamoxifen (75mg / kg) (Sigma Aldrich) as a single IP injection daily for 5 consecutive days using a 20 mg / mL stock of tamoxifen in com oil. This causes depletion of SIRPa in microglia and infiltrating monocytes / MG since both cell types express Cx3crlcreERT2.12-61-62Experiments were conducted 1 week after the last dose of tamoxifen when myeloid Cre activity is high and tamoxifen is eliminated from the circulation since it is a neuroprotective agent.63,64

[0135] Optic nerve crush model

[0136] Mice were anesthetized with isoflurane and treated with a single subcutaneous injection of buprenorphine extended-release (3.25 mg / kg) for analgesia. To induce optic nerve crush (ONC) injury, the left optic nerve was exposed and crushed for 3 seconds using a self-closing forceps (Dumont, #N7 forceps) as we previously described (PMID: 34780773, PMID: 30618589). The contralateral eye was utilized as non-injury sham.

[0137] Antibody Treatment

[0138] To investigate the relationship between TNF- a and CD47 expression, WT mice received 1 pL intravitreal injection of anti-TNF- a (cone. 9.81 mg / ml, BioXCell) on day 3 post ONC injury. For CD47 / SIRPa antibody treatment experiments, wild-type C57BL / 6 mice received 1 pL intravitreal injection of either SIRPa (cone. , BioXCell), CD47 (cone. 8.31 mg / ml, BioXCell), or IgG (cone. 10.22 mg / ml, BioXCell) antibodies on day 4 post-ONC. Injections were given via a microinjection syringe pump (World Precision Instruments).

[0139] Antibodies labeling

[0140] For antibody penetration studies, SIRPa antibodies were labeled with rhodamine (Thermo Scientific) and injected at day 4 post-ONC whereas saline injection was used as control. Mice were sacrificed at days 4 through 7. Eyeballs were collected and fixed in 4% paraformaldehyde, and retinas were dissected into flat mounts on slides. Zeiss LSM 880 with Airyscan confocal microscope was used for fluorescence imaging.

[0141] Western Blot

[0142] For in vivo experiments, retinas were collected, snap-frozen, and stored at -80 for later analysis. Retinas were homogenized and suspended in RIPA lysis buffer (Thermo Scientific) and centrifuged at 20,000 x g. Protein was then estimated using Pierce™ Rapid Gold BCA Protein Assay Kit (Thermos Scientific). For in vitro experiments, cells were washed twice with PBS before collection in RIPA lysis buffer. Samples’ lysates were run on SDS-PAGE and transferred to nitrocellulose membranes (BioRad). Primary antibodies used were anti-TNF-a (Abeam), anti- CD47 ( R&D systems)

[0143] Immunofluorescent labeling

[0144] Eyeballs were collected and fixed in 4% paraformaldehyde overnight at 4 °C. After fixation, they were transferred to phosphate-buffered saline (PBS) and stored at 4 °C until analysis. Images were taken using Zeiss LSM 880 with Airyscan confocal microscope. Neurodegeneration was assessed at 14 days post ONC. Retinas were dissected from eyeballs, and neurons were immunolabeled using the neuronal marker anti-NeuN (Millipore Sigma) and flat-mounted. Images were taken from the retinal mid-periphery and neurodegeneration was analyzed as NeuN+ area in the ganglion cell layer (GCL) using hnageJ software. Eyeballs were collected on day 4 post-ONC for the anti-TNFa treatment groups. CD47 levels were analyzed using anti-CD47 (R&D systems).

[0145] Efferocytosis Index

[0146] For the efferocytosis indexes, eyeballs were collected and fixed in 4% paraformaldehyde overnight at 4 °C on day 5 post-ONC. The efferocytosis index was assessed using Click-iT™ Plus TUNEL Assay (Invitrogen) and anti-Iba-1 (FUJIFILM Wako). Co-localization of TUNEL and IBA-1 positive cells was manually counted.

[0147] Retinal Optical Coherence Tomography (OCT)

[0148] Pupils were dilated using 1% tropicamide (Akron Pharmaceuticals, IL) and mice were anesthetized with ketamine / xylazine prior to optical coherence tomography (OCT, Bioptigen Inc., Durham, NC) imaging to assess retinal thickness between groups (rectangular volume mode - 3 frames / scan, 1000 A scan / B scan x 100 B scan, 1.4 mm x 1.4 mm). Images were analyzed using InVivoVue™ Diver 2.4 software (Bioptigen Inc., Durham, NC).

[0149] Pattern Electroretinography (PERG)

[0150] PERG on Celeris electroretinogram system (Diagnosys LLC, Cambridge, UK) was used to assess retinal functional differences between groups. Mice were dark adapted overnight before PERG assessment at 14 days post ONC. Pupils were dilated using 1% tropicamide (Akron Pharmaceuticals, IL) and mice were anesthetized with ketamine / xylazine prior to testing. PERG was performed under dim red lighting. Eyes were assessed with one pattern stimulator used for the test eye and a full field stimulator used as a reference electrode for the fellow eye. The amplitude of Pl, N2, and |P 1 -N2| was used to assess retinal functionality.

[0151] Cell Culture

[0152] All cells and retinal explants were incubated in a humidified incubator at 37 °C with 5% CO2.

[0153] Bone Marrow-Derived Macrophages (BMDM)

[0154] Bone marrow cells were collected in 20 ml of sterile PBS from femurs and tibias of C57BL / 6 mice using 27-guage needle to flush out cells. Cells were then resuspended in M differentiation media, (Dulbecco’s modified Eagle’s medium, DMEM, high glucose, (Gibco; Thermo Fisher, NY) containing 20% FBS (Gibco; Thermo Fisher, NY) 20% L929 conditioned media, and 1% penicillin-streptomycin (pen-strep)) and plated on uncoated 100-mm dishes for differentiation as previously described (PMID: 30254218).

[0155] Retinal Explants

[0156] C57BL / 6 mice were anesthetized and sacrificed via cervical dislocation. Eyecups were harvested after sacrifice and retinas were immediately harvested from eyecups in ice-cold HBSS containing 1% penicillin-streptomycin. Retina was placed face up in transwells inserts (Thermo Scientific) in a 24-well plate supplied with Neurobasal-A medium (Invitrogen) with 2% B27 (Invitrogen), 1% N2 (Invitrogen), and 2mM GlutaMAX (Invitrogen).

[0157] Statistical Analysis

[0158] Statistical analyses were prepared using GraphPad Prism 10 software and data were presented as mean ± standard error of the mean (SEM). A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using the student’s t-test (for two-group comparisons), one or two-way analysis of variance (ANOVA) with Tukey’s post hoc test (for comparison of multiple groups).

[0159] Results:

[0160] CD47 and TNF-a are upregulated in the retina after ONC injury

[0161] After ONC injury, cells in the ganglion cell layer become apoptotic and immune cells are recruited to the retina. We first conducted a time course of apoptotic cell induction after crush using TUNEL labeling of WT retina cross-sections which showed peak accumulation of apoptitic cells with 4-5 days after ONC. (Fig. 11A). Flatmount immunolabeling showed proliferation / infiltration of Iba-1+ myeloid cells starting at day 2 and persisting till day 7. Immune cells recruitment to apoptotic cells facilitates their clearance through a reparative process called efferocytosis which is tightly regulated by ‘eat me’ and ‘don’t eat me’ signals. The ‘don’t eat me’ signal, CD47, has been found to be upregulated in certain pathological conditions, including cancers and atherosclerosis.3,4Here we investigated the role of CD47 and its ligand SIRPa in traumatic optic neuropathy. To do this, we first performed ONC on C57BL6 / J WT mice (contralateral eye used as a non-injured sham). Western blot of retinal lysates revealed upregulation of CD47 on day 4 post-ONC through day 10 (Fig.HC). Immunolabeling of retina sections revealed an increase in CD47 levels in the retinal ganglion cell layer, which is the layer most susceptible to injury in this TON model (Fig. HD).

[0162] Previous research identified the cytokine tumor necrosis factor-alpha (TNF-a) as a major inducer of CD47.18Interestingly, we also found sustained upregulation of TNF-a in retinal lysates, starting at day 3 post-ONC injury and continuing through day 14 (Fig. HE).

[0163] TNF-a mediates CD47 upregulation after ONC injury.

[0164] Since the upregulation of TNF-a occurs 24 hours prior to CD47 upregulation, we conducted a series of experiments to determine whether TNF-a plays a role in the upregulation of CD47 in retinal ganglion cells. To do this, we incubated WT retinal explants with increasing concentrations of recombinant TNF-a (0, 20, and 50 ng / ml) for 24 hours. Explants were then processed for Western blot and immunolabeling. Western blotting on retinal lysates showed significant upregulation of CD47 with TNF-a (20 and 50 ng / ml) treatment (Fig. 12A-B). To determine if TNF-mediated CD47 upregulation is associated with retinal ganglion cell death we immunolabled retinal explant flat-mount with NeuN (neuronal maker) which showed a neurodegeneration trend at 20 ng / ml and significant degeneration with 50 ng / ml recombinant TNF-a treatment, *p<0.05 (Fig. 12C-D). This suggested to us that TNF-mediated CD47 upregulation precedes TNF-induced neuronal death.

[0165] To further determine the contribution of TNF in CD47 upregulation in vivo, we administered anti-TNF-a antibodies intravitreally to WT mice after ONC. Since we first saw the upregulation of TNF-a on day 3 post-ONC, we chose day 3 to treat the mice with anti-TNF-a 1 pL intravitreal injection in the injured and contralateral sham eye. Retinas were collected for Western blot and IHC. Western blot analysis of whole retinal lysates revealed no significant difference in CD47 expression between groups. However, treatment with anti-TNF-a resulted in downregulation of TNF-a, suggesting successful neutralization. (Fig 12G-I) Immunolabeling revealed that treatment with anti-TNF-a resulted in downregulation of CD47 in the retinal ganglion cell layer after ONC.

[0166] (Fig 12E-F).

[0167] CD47 Neutralization Improves Efferocytosis in TON

[0168] To investigate the role of the CD47 / SIRPa in efferocytosis and injury resolution after ONC, we tested the therapeutic potential of CD47 and SIRPa neutralizing antibodies with anti- IgG used as control. Since CD47 is upregulated at day 4, we employed a delayed intravitreal administration of the neutralizing antibodies at day 4. To confirm that antibodies were retained in the retina after treatment, we injected rhodamine -labeled anti-SIRPa on day 4 and collected post- ONC. Antibodies were retained in the retina through day 7 (Fig.l4A). On day 5 post-ONC, retinas were collected for efferocytosis index (El). El was determined based on the colocalization of TUNEL (an apoptosis marker) and IBA-1 (a myeloid marker). Anti-IgG has increased apoptotic cells present. Anti-CD47 treatment results in an increase in macrophage-associated apoptotic cells. Treatment with anti-CD47 improves macrophage-mediated clearance of dead cells in the retina following ONC injury (Fig 13A-C)(n=8-9 unpaired t-test *p<0.05 vs. anti-IgG treated). At day 14, IHC showed a downward trend in CD45+ and CD68+ cells with anti-CD47 treatment but was not significant.

[0169] CD47 Blockade Attenuates Neurodegeneration and Improves Retinal Function

[0170] To investigate the therapeutic effect of CD47 and SIRPa neutralizing antibodies (anti-IgG used as control) WT mice received 1 ul intravitreal injection of neutralizing antibodies at concentration discussed above on day 4 post ONC injury. On day 14 post-ONC injury, retinal thickness was determined with OCT, retinal function with PERG, and retinas were then collected for IHC. NueN staining revealed attenuated neurodegeneration with anti-CD47 treatment (Fig. 14B-C). Anti-SIRPa treatment resulted in neuroprotective trend but not significant (Fig. 14D-E).

[0171] Day 14 OCT imaging further revealed rescue of retinal ganglion cell complex thickness with anti-CD47 but not anti-SIRPa treatment (Fig 15. A-C). (n=5-7 unpaired t test *p<0.05 vs. anti-IgG treated). Retinal ganglion cell functionality was also assessed at the second week before sacrifice using PERG. Both anti-CD47 and anti-SIRPa showed improved functionality trend for Pl and N2 amplitudes (Fig. 15C, D, E, G, H). However, results were not significant. Importantly, anti-CD47 treatment showed overall improved retinal ganglion cell function (Fig. 15F). Anti- SIRPa treatment resulted in improved retinal function trend but not results were not significant (Fig. 151).

[0172] Myeloid-specific SIRPa Deletion Improves Retinal Function

[0173] SIRPa mice (and SIRPaf / fmice) were used to investigate further the role of CD47 / SIRPa axis in TON and to confirm anti-SIRPa experiments shown above. For all experiments, SIRPa mice and their SIRPaf / flittermates received tamoxifen injections for sequential 5 days. Since tamoxifen has been shown to be neuroprotective experiments began one week after tamoxifen injections. On day 14 post-ONC injury, myeloid deletion of SIRPa resulted neuroprotective trend for NueN staining and OCT. However, results were not significant (Fig 16 A-D). Interestingly, functionality, measured via PERG, showed significant improvement in SIRPa RGC functionality (Fig. 16E-F).

[0174] Discussion:

[0175] In this study we reported four major findings: First, we have found upregulation of the ‘don’t eat me’ signal CD47 in the inner retina during the subacute phase of the ONC injury. Second, we have identified TNF-a as the mediator of CD47 upregulation. Third, our study provides the first evidence of neuronal protection and improved retinal function with blockade of the CD47 / SIRPa axis after ONC injury. Fourth, this protection was mediated by enhanced efferocytosis of apoptotic cells by myeloid cells.

[0176] CD47 is a myeloid cell immune checkpoint that inhibits efferocytosis (the removal of apoptotic cells) by macrophages and other phagocytic cells. It achieves this by interacting with the SIRPa receptor on these cells, thus preventing them from engulfing and removing cells that express CD47. While CD47 is expressed on almost all healthy cells to help them be recognized as "self by the immune system, its upregulation has been linked to pathological conditions. CD47 upregulation on apoptotic cells has been linked to disease progression in cancer and atherosclerosis through impaired efferocytosis. Our data is the first to implicate CD47 upregulation in the pathogenesis of traumatic optic neuropathy. Since TNF-a upregulates CD47 in many cell types, we investigated its regulation of CD47 after ONC. Compelling evidence, in vivo and ex vivo, implicated TNF-a as a regulator of CD47 after ONC. TNF-a upregulates CD47 primarily in the retinal ganglion cell layer after ONC.

[0177] TNF-a plays a complex role in RGC injury and survival which could be explained by the opposing effects of its receptors, TNFR1 and TNFR2. TNFR1 is known to induce apoptosis, and an earlier study showed that deletion of TNFR1 decreased neurodegeneration after ONC; however, this effect was only prominent after 2 weeks from injury. TNFR1 appears to mediate similar RGC neurodegeneration following retinal ischemia, while TNFR2 plays a protective role. Inhibition of TNF with etanercept, which blocks both soluble and membrane -bound TNF-a, increased RGC survival after ONC. Interestingly, a study using TNF-a KO mice showed enhanced neurodegeneration in these mice after crush suggesting a protective role of TNF-a. The authors confirmed this protective role of TNF-a using intravitreal pretreatment with exogenous TNF-a. However, TNF-a treatment was protective only when given 5 days prior to ONC and TNF-a treatment at 7 days after crush did not affect neuronal survival. However, we have found that recombinant TNF-a led to worsened RGC neurodegeneration in a dose-dependent manner in retinal explants. Interestingly, TNF-a upregulated CD47 in a sub-lethal dose suggesting that the induction of CD47 by TNF-a is independent from its pro-apoptotic effects.

[0178] Improving efferocytosis after neuronal injury has been increasingly studied in recent years for its potential therapeutic effect. Enhancing efferocytosis has been shown to improve traumatic brain injury outcomes. Here, we provide the first evidence that efferocytosis plays a reparative role in traumatic optic neuropathy. We have found that blockade of the “Don’t eat me” signal, CD47, with neutralizing antibodies results in increased efferocytic efficiency, determined via colocalization of TUNEL+ cells with IBA-1 positive cells. Furthermore, injured retinas treated with anti-CD47 had a decrease in apoptotic cells, possibly due to the increased interaction between dying cells and myeloid cells.

[0179] Neutralization of CD47 ameliorated neurodegeneration and rescued retinal ganglion cell function after ONC injury. This finding complements other studies which have also found blockade of the CD47 / SIRPa axis in stroke and uveitis improves neuronal cell survival and functional outcomes. Interestingly, even though there was a neuroprotective trend, we did not observe a significant increase in NeuN+ neurons or retinal function with antibody blockade of SIRPa. CD47 may be a better therapeutic target than SIRPa in ONC due to more than just phagocytosis. For example, when thrombospondin- 1 (TSP-1) binds to CD47 it blocks nitric oxide signaling, which typically promotes vasodilation and cell survival. Therefore, blockade of CD47 would also block CD47 / TSP- 1 signaling, possibly leading to increased blood and oxygen supply to the cite of injury resulting in improved cellular metabolism, function, and survival. Additional studies are needed to further define these pathways in TON. Another possible explanation of the discrepancy between CD47 and SIRPa neutralization is that expression of SIRPa on neurons has been shown to counterintuitively enhance microglial phagocytosis during retinal development through acting as a decoy receptor to prevent microglial SIRPa-CD47 interaction. To dissect the role of myeloid cell SIRPa, we generated and utilized a myeloid specific knockout of SIRPa to further investigate the inhibition of SIRPa in ONC. Surprisingly, we found that while myeloid deletion of SIRPa only showed a protective trend in structural markers, it significantly improved RGC function. This indicates that myeloid SIRPa may impair RGC function even before complete cell death. In summary, our studies highlight the therapeutic potential of improving efferocytosis with the blockade of the CD47-SIRPa axis in ONC, leading to neuroprotection and improved retinal function. Since CD47-SIRPa are already in clinical testing for certain cancers this will facilitate their translation to the clinic for traumatic optic neuropathy. The delayed treatment employed in this study suggests a wide window for intervention with these antibodies.

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Claims

CLAIMSWhat is claimed:

1. A method of treating a traumatic or ischemic central nervous system (CNS) injury, the method comprising administering an antagonist of the interaction between CD47 and SIRPa.

2. The method of claim 1, wherein the antagonist is selected from the group consisting of an antibody, a small molecule, an oligonucleotide, a recombinant protein, and an aptamer.

3. The method of claim 2, wherein the antagonist comprises an anti-CD47 antibody.

4. The method of claim 2, wherein the antagonist comprises an anti-SIRPa antibody.

5. The method of claim 2, wherein the antibody is selected from the group consisting ofTTI-621, TTI-622, Hu5F9-G4, ALX148 and CC-95251.

6. The method of claim 2, wherein the small molecule is selected from the group consisting of NCGC00138783, PEP-20 and D4-2.

7. The method of any one of the preceding claims, wherein the traumatic or ischemic CNS injury is selected from the group consisting of stroke, ischemic retinopathies such as diabetic retinopathy, retinal artery or vein occlusion, retinopathy of prematurity, spinal cord injury and traumatic brain injury.

8. The method of claim 7, wherein the stroke is ischemic stroke.

9. The method of any one of the preceding claims, wherein the antagonist is administered within 3 hours of the CNS injury.

10. The method of any one of the preceding claims, wherein the treatment reduces infarction area, swelling, or recovery time.

11. The method of any one of the preceding claims, wherein the treatment increases cognitive recovery and / or motor function recovery.

12. A composition comprising an antagonist of the CD47 SIRPa interaction for use in preparation of a medicament for treatment of ischemic stroke.

13. A method of treating traumatic optic neuropathy, the method comprising administering an antagonist of the interaction between CD47 and SIRPa.

14. The method of claim 13, wherein the treatment increases or improves visual acuity, color vision, visual field deficit, axonal regeneration or neuroprotection.

15. The method of claim 13, wherein the treatment decreases optic atrophy.

16. A method of increasing efferocytosis after a traumatic or ischemic central nervous system (CNS) injury, the method comprising administering an antagonist of the interaction between CD47 and SIRPa.

17. The method of any one of claims 13-16, wherein the antagonist is selected from the group consisting of an antibody, a small molecule, an oligonucleotide, a recombinant protein, and an aptamer.

18. The method of claim 17, wherein the antagonist comprises an anti-CD47 antibody.

19. The method of claim 17, wherein the antagonist comprises an anti-SIRPa antibody.

20. The method of claim 17, wherein the antibody is selected from the group consisting ofTTI-621, TTI-622, Hu5F9-G4, ALX148 and CC-95251.

21. The method of claim 17, wherein the small molecule is selected from the group consisting ofNCGC00138783, PEP-20 and D4-2.

Images