Compositions and methods for Anti-inflammatory peptides

Abstract

Compositions of anti-inflammatory peptides and methods of use thereof are provided. The peptides include N-terminal, palmitoylated peptides covering / derived from a coiled-coil region of CRACR2A. Methods include reducing inflammation and / or inhibiting a STIM1-CRACR2A interaction in a subject in need thereof by administering a composition of the present disclosure. Exemplary compositions include palmitoylated peptides having between about 10 and 20 amino acids.

Description

[0001] Docket No.: 021025 / WO

[0002] COMPOSITIONS AND METHODS FOR ANTI-INFLAMMATORY PEPTIDES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 713,364 filed 29 October 2024, which is incorporated herein by reference in its entirety.

[0005] STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under HL130028 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] MATERIAL INCORPORATED BY REFERENCE

[0008] The Sequence Listing, which is a part of the present disclosure, includes a computer- readable form comprising nucleotide and / or amino acid sequences of the present invention (file name “021025-W0_2025-10-29_Sequence-Listing” created on 29 October 2025; 34,441 bytes). The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

[0009] FIELD

[0010] The present disclosure generally relates to anti-inflammatory peptides and their use in mitigating inflammation and tissue injury.

[0011] BACKGROUND

[0012] While neutrophils are important for innate immune responses, their excessive recruitment to sites of inflammation causes tissue damage. Neutrophil recruitment on activated endothelial cells (ECs) is a sequential process, including rolling, arrest, crawling, and transmigration, mediated by the interactions between neutrophil and EC adhesion molecules. After neutrophil rolling, mediated by the interaction between selectins and their ligands, neutrophils adhere and crawl on ECs, which are mainly controlled by aL[32 and aM[32 integrin, respectively. The final step of neutrophil transmigration requires various molecules, including (32 integrin, CD31 , and junctional adhesion molecules. Although neutrophil surface receptors required for neutrophil recruitment have been well identified, how their function is regulated remains poorly understood. Docket No.: 021025 / WO

[0013] As a large family of small GTPases, Rab GTPases are involved in intracellular vesicle trafficking in numerous cell types and contribute to the pathology of cancer, neurodegenerative diseases, diabetes, and immune diseases. Among them, Ca2+ release- activated Ca2+ channel regulator 2A (CRACR2A, also known as Rab46) exists in both long (CRACR2A-L, around 90-kDa) and short (CRACR2A-S, around 45-kDa) forms. CRACR2A- L contains two EF-hands, a coiled-coil domain, a Pro-rich domain, a Rab GTPase domain, and a C-terminal prenylation site, whereas CRACR2A-S only contains two EF-hands and a coiled-coil domain. In T cells, both CRACR2A-L and CRACR2A-S promote store-operated Ca2+ entry (SOCE) and activate the Ca2+-nuclear factor of activated T-cells (NFAT) signaling pathway. Importantly, a recent study identified a patient with biallelic mutations in the CRACR2A gene, who exhibited reduced cytokine production and SOCE in T cells with the development of an immunodeficiency disorder. However, the role of neutrophil CRACR2A in inflammation is unknown.

[0014] The CRACR2A-STIM1 -Orai1 complex is critical for increasing cytosolic Ca2+ levels in stimulated T-cells. Some inhibitors are being developed to block STIM1 or Orail as antiinflammatory diseases. However, gene deletion or mutations in STIM1 or Orail in humans is linked to immunodeficiency, leading to death in patients. Consistently, deletion of STIM1 or Orail in neutrophils markedly diminishes the production of reactive oxygen species (ROS) after agonist stimulation. Since ROS are critical for neutrophil innate immune function, this result corroborates the clinical findings that targeting STIM1 or Orail may impair innate immunity. Therefore, alternative therapeutic targets are needed.

[0015] BRIEF DESCRIPTION OF THE DISCLOSURE

[0016] Among the various aspects of the present disclosure is the provision of antiinflammatory peptide compositions and methods of use thereof.

[0017] In accordance with an aspect of the present disclosure, an anti-inflammatory composition is provided. The anti-inflammatory composition comprises: one or more peptides derived from a coiled-coil region of CRACR2A. In some embodiments, at least one of the one or more peptides is palmitoylated. In some embodiments, each of the one or more peptides is not less than a 10-mer peptide and is not more than a 50-mer peptide. In some embodiments, at least one of the one or more peptides is selected from SEQ ID NO: 16-32. Docket No.: 021025 / WO

[0018] In some embodiments, at least one of the one or more peptides is SEQ ID NO: 17. In some embodiments, at least one of the one or more peptides is selected from SEQ ID NO: 33-38.

[0019] In accordance with another aspect of the present disclosure, a method of reducing inflammation in a subject in need thereof is provided. The method comprises: administering to the subject a therapeutically effective amount of an anti-inflammatory composition comprising one or more peptides derived from a coiled-coil region of CRACR2A. In some embodiments, the subject has sterile inflammation, sterile inflammation caused by ischemic stroke, sterile inflammation caused by vasculitis, or sterile inflammation caused by an autoimmune disease. In some embodiments, at least one of the one or more peptides is palmitoylated. In some embodiments, each of the one or more peptides is not less than a 10- mer peptide and is not more than a 50-mer peptide. In some embodiments, at least one of the one or more peptides is selected from SEQ ID NO: 16-32. In some embodiments, at least one of the one or more peptides is selected from SEQ ID NO: 33-38. In some embodiments, the anti-inflammatory composition is administered at a concentration of at least 10 pM.

[0020] In accordance with a further aspect of the present disclosure, a method of blocking a STIM1 -CRACR2A interaction in a subject in need thereof is provided. The method comprises: administering to the subject a therapeutically effective amount of a composition comprising one or more peptides derived from a coiled-coil region of CRACR2A. In some embodiments, blocking the STIM1-CRACR2A interaction in the subject reduces inflammation, reduces neutrophil migration to a site of inflammation, reduces neutrophil adhesion to endothelial cells (EC), inhibits aM[32 integrin activation, and / or blocks Ca2+ mobilization. In some embodiments, at least one of the one or more peptides is selected from SEQ ID NO: 16-32. In some embodiments, at least one of the one or more peptides is selected from SEQ ID NO: 33-38. In some embodiments, the composition is administered at a concentration of at least 10 pM. In some embodiments, the subject has sterile inflammation, sterile inflammation caused by ischemic stroke, sterile inflammation caused by vasculitis, or sterile inflammation caused by an autoimmune disease.

[0021] Other objects and features will be in part apparent and in part pointed out hereinafter. Docket No.: 021025 / WO

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Those of skill in the art will understand that the drawings described herein are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0024] FIG. 1 A is a set of images of (top) a PCR gel of WT (+ / +) control, cracr2a heterozygous conditional knockout (CKO; fl / +), and CKO KO (fl / fl) mice and (bottom) a PCR gel of WT (-) and Lyz-cre (+) mice.

[0025] FIG. 1 B is a representative western blot of lysates of mouse (mN; C57BL / 6) and human neutrophils (hN) showing long-form and short-form expression of CRACR2A (n=3).

[0026] FIG. 1 C is a representative set of western blots showing expression of the indicate protein in WT and CKO neutrophil (left), monocyte (middle) and platelet (right) (n=3).

[0027] FIG. 1 D is a schematic showing the experimental setup wherein WT and cracr2a CKO mice are treated with an intravenous injection (I.V.) of anti-CD31 and anti-Ly-6G antibodies and an intraperitoneal injection (I.P.) of TNF-a, followed by intravital microscopy (IVM) of ear venules.

[0028] FIG. 1 E is a set of representative IVM images of WT (left) and cracr2a CKO (right) ear venules showing expression of CD31 (dark grey) and Ly6G (light grey). Arrowheads indicate transmigrated and transmigrating (TEM) neutrophils. Bar = 50 pm.

[0029] FIG. 1 F is a graph showing the number of rolling neutrophils in WT and CKO mice after I.V injection of anti-CD31 and anti-Ly-6G antibodies and I.P. injection of TNF-a. *p < 0.05.

[0030] FIG. 1 G is a graph showing the number of adherent neutrophils in WT and CKO mice after I.V injection of anti-CD31 and anti-Ly-6G antibodies and I.P. injection of TNF-a. ****p < 0.0001.

[0031] FIG. 1 H is a graph showing the number of crawling neutrophils in WT and CKO mice after I.V injection of anti-CD31 and anti-Ly-6G antibodies and I.P. injection of TNF-a. **p < 0.01.

[0032] FIG. 11 is a graph showing the number of TEM neutrophils in WT and CKO mice after I.V injection of anti-CD31 and anti-Ly-6G antibodies and I.P. injection of TNF-a. ****p < 0.0001. Docket No.: 021025 / WO

[0033] FIG. 1 J is a schematic showing the experimental setup wherein WT and CKO mice were treated with an I.V injection of anti-Ly-6G and an intrascrotal injection (I.S.) of TNF-a, followed by IVM of cremaster venules.

[0034] FIG. 1 K is a set of representative IVM images of WT (left) and cracr2a CKO (right) cremaster venules showing Ly6G neutrophils (bottom; green) and merged (top) images.

[0035] FIG. 1 L is a graph showing the number of rolling neutrophils in WT and CKO mice after an I.V injection of anti-Ly-6G and an I.S. injection of TNF-a.

[0036] FIG. 1 M is a graph showing the number of adherent neutrophils in WT and CKO mice after an I.V injection of anti-Ly-6G and an I.S. injection of TNF-a. *p < 0.05.

[0037] FIG. 1 N is a graph showing the number of crawling neutrophils in WT and CKO mice after an I.V injection of anti-Ly-6G and an I.S. injection of TNF-a. ****p < 0.0001 .

[0038] FIG. 10 is a graph showing the number of TEM neutrophils in WT and CKO mice after an I.V injection of anti-Ly-6G and an I.S. injection of TNF-a.

[0039] FIG. 2A is a graph quantifying, from flow cytometry, the surface amount of aM[32 integrin on neutrophils treated with 20 ng / ml TNF-a, 5 pM fMLP, or 0.5 pM A23187 in the presence of 1 mM CaCl2. Student’s t-test; **P < 0.01 , ***P < 0.001

[0040] FIG. 2B is a graph quantifying, from flow cytometry, the binding of Alexa Fluor 488- conjugated fibrinogen (A488-FG) to neutrophils treated with 20 ng / ml TNF-a, 5 pM fMLP, or 0.5 pM A23187 in the presence of 1 mM CaCL. Student’s t-test; *P < 0.05, **P < 0.01 .

[0041] FIG. 2C is a graph showing fibrinogen (FIG. 2B) normalized to the surface level of aM|32 integrin (FIG. 2A). Student’s t-test; *P < 0.05.

[0042] FIG. 2D is a representative western blot (top) and corresponding graph (bottom) of WT and cracr2a-deficient neutrophils (CKO) treated with 5 pM fMLP for 10 minutes. The cell lysates were immunoprecipitated with anti-[32 antibodies and immunoblotted with anti-talin1 or anti-|32 antibodies (top), followed by densitometry (bottom). Student’s t-test; *P < 0.05, **P < 0.01.

[0043] FIG. 2E is a representative western blot (top) and corresponding graph (bottom) of WT neutrophils were treated with or without 5 pM fMLP for 5 minutes. The lysates were immunoprecipitated with anti-CRACR2A antibodies and immunoblotted with anti-STIM1 or anti-CRACR2A antibodies (top), followed by densitometry (bottom). Tukey’s test; **P < 0.01.

[0044] FIG. 2F is a graph showing representative traces of WT (solid line) and cracr2a- deficient (dashed line) neutrophils incubated with Fura2 (red) or without Fura2 (black). Ca2+ Docket No.: 021025 / WO mobilization was measured after stimulation with 5 pM fMLP in the presence of 1 mM CaCI? (n=3-4; *P < 0.05, **P < 0.01 ).

[0045] FIG. 2G is a graph showing representative traces of WT (solid line) and cracr2a- deficient (dashed line) neutrophils incubated with Fura2 (red) or without Fura2 (black). Ca2+mobilization was measured after stimulation with 0.5 pM A23187 in the presence of 1 mM CaCI2(n=3-4).

[0046] FIG. 2H is a graph showing representative traces of WT (solid line) and cracr2a- deficient (dashed line) neutrophils incubated with Fura2 (red) or without Fura2 (black). Ca2+mobilization was measured after stimulation with 10 pM thapsigargin (TG) in the presence of 1 mM CaCI2(n=3-4).

[0047] FIG. 2I is a graph quantifying the area under the curve (AUC) of Ca2+mobilization of WT and CKO neutrophils treated with fMLP, A32187, or TG (from FIG. 2F, FIG. 2G, and FIG. 2H). n=3-4; Student’s t-test; *P < 0.05, **P < 0.01 .

[0048] FIG. 2J is a set of representative images over time (2, 5, and 10 minutes) of WT (left) and cracr2a CKO (right) neutrophils incubated with fMLP (bottom) or unstimulated (top).

[0049] FIG. 2K is a graph showing the area of adherent WT and cracr2a-deficient neutrophils labeled with calcein-AM and treated with or without 5 pM fMLP. The data represent the mean ± SD; Student’s t-test; *P < 0.05.

[0050] FIG. 2L is a graph showing the area of spreading WT and cracr2a-deficient neutrophils labeled with calcein-AM and treated with or without 5 pM fMLP. The data represent the mean ± SD (n = 4 and 105-157 cells in 4 independent experiments); Student’s t-test; *** P < 0.001.

[0051] FIG. 2M is a graph of the area of spreading WT and cracr2a-deficient neutrophils plated on a chamber slide coated with FG and incubated in the presence or absence of 5 pM fMLP. The data represent the median values (n = 62-76 cells in 4 independent experiments); Mann-Whitney U test; ****P < 0.0001.

[0052] FIG. 2N is a graph showing the Pearson coefficient of the location between F-actin and p-MLC from WT and cracr2a-deficient neutrophils plated on a chamber slide coated with FG and incubated in the presence or absence of 5 pM fMLP. The data represent the median values (n = 62-76 cells in 4 independent experiments) Mann-Whitney U test; ****P < 0.0001.

[0053] FIG. 20 is a set of immunofluorescent images of WT and cracr2a-deficient neutrophils plated on a chamber slide coated with FG. After incubation for 10 minutes in the presence or absence of 5 pM fMLP, adherent neutrophils were fixed and incubated with anti-p-MLC Docket No.: 021025 / WO antibodies. Then, cells were incubated with Alexa Fluor 488-conjugated phalloidin and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies, followed by confocal microscopy (left, merge; left middle, p-MLC; right middle, F-actin; right, nucleus).

[0054] FIG. 3A is a schematic showing a strategy of CRACR2A deletion in HL-60 by CRISPR / Cas9, see also SEQ ID NO: 14 and SEQ ID NO: 15.

[0055] FIG. 3B is a set of flow cytometry plots and a corresponding graph showing the purity of WT (left) and CRACR2A KO (right) dHL-60 cells.

[0056] FIG. 3C is a representative image of a western blot of lysates of WT (left) and CRACR2A KO (right) dHL-60 cells (n = 3).

[0057] FIG. 3D is a graph showing the surface level of total aM[32 in WT and CRACR2A KO dHL-60 cells treated with 5 pM fMLP or 0.25 pM A23187. Student’s t-test; *P < 0.05, **P < 0.01 , ***P < 0.001.

[0058] FIG. 3E is a graph showing the surface level of activated aM[32 in WT and CRACR2A KO dHL-60 cells treated with 5 pM fMLP or 0.25 pM A23187. Student’s t-test; *P < 0.05, ***P

[0059] < 0.001.

[0060] FIG. 3F is a graph showing the surface level of activated (32 integrin in WT and CRACR2A KO dHL-60 cells treated with 5 pM fMLP or 0.25 pM A23187. Student’s t-test; *P

[0061] < 0.05, **P < 0.01.

[0062] FIG. 3G is a plot showing representative traces of cytosolic Ca2+levels in WT and CRACR2A KO dHL-60 after stimulation with 5 pM fMLP in the presence of 1 mM CaCL.

[0063] FIG. 3H is a plot showing representative traces of cytosolic Ca2+levels in WT and CRACR2A KO dHL-60 after stimulation with 0.25 pM A23187 in the presence of 1 mM CaCh

[0064] FIG. 3I is a plot showing representative traces of cytosolic Ca2+levels in WT and CRACR2A KO dHL-60 after stimulation with 5 pM thapsigargin (TG) in the presence of 1 mM CaCI2.

[0065] FIG. 3J is a graph quantifying (area under the curve (AUC)) Ca2+mobilization in WT and CRACR2A KO dHL-60 after stimulation with 5 pM fMLP, 0.25 pM A23187, or 5 pM thapsigargin (TG) in the presence of 1 mM CaCL. Student’s t-test; *P < 0.05, **P < 0.01.

[0066] FIG. 3K is a representative blot of lysates of WT and CRACR2A KO cells expressing mCherry, mCherry-tagged CRACR2A-L- or CRACR2A-S-overexpressed dHL60 cells (n = 3). Docket No.: 021025 / WO

[0067] FIG. 3L is a graph of Ca2+mobilization in WT and CRACR2A dHL-60 cells expressing control vector, CRACR2A-L- or CRACR2A-S after stimulation with 5 pM fMLP in the presence of 1 mM CaCl2. ANOVA; ***P < 0.001 , and ****P < 0.0001 .

[0068] FIG. 3M is a graph of Ca2+mobilization in WT and CRACR2A dHL-60 cells expressing control vector, CRACR2A-L- or CRACR2A-S after stimulation with 0.25 pM A23187 in the presence of 1 mM CaCl2. ANOVA; **P < 0.01 and ****P < 0.0001.

[0069] FIG. 3N is a graph of Ca2+mobilization in WT and CRACR2A dHL-60 cells expressing control vector, CRACR2A-L- or CRACR2A-S after stimulation with 5 pM TG in the presence of 1 mM CaCI2. ANOVA; ***P < 0.001 .

[0070] FIG. 30 is a graph showing the surface level of total aM[32 in control and CRACR2A- L- or CRACR2A-S-overexpressed WT and KO dHL-60 cells after stimulation with 5 pM fMLP or 0.25 pM A23187 in the presence of 1 mM CaCb and 1 mM MgCL. The data represent the mean ± SD (n = 4). ANOVA; **P < 0.01 , ***P < 0.001 .

[0071] FIG. 3P is a graph showing the surface level of activated aM[32 in control and CRACR2A-L- or CRACR2A-S-overexpressed WT and KO dHL-60 cells after stimulation with 5 pM fMLP or 0.25 pM A23187 in the presence of 1 mM CaCL and 1 mM MgCL. The data represent the mean ± SD (n = 4). ANOVA; **P < 0.01 , ***P < 0.001 .

[0072] FIG. 3Q is a graph showing the surface level of activated [32 integrin in control and CRACR2A-L- or CRACR2A-S-overexpressed WT and KO dHL-60 cells after stimulation with 5 pM fMLP or 0.25 pM A23187 in the presence of 1 mM CaCL and 1 mM MgCL. The data represent the mean ± SD (n = 4). ANOVA; *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0073] FIG. 4A is a graph showing the viability of mouse Ly-6G neutrophils treated with peptides 1-9. The N-terminal palmitoylated peptides (eight 20-mers (P1 -P8) and one 16-mer (P9)) covering the coiled-coil region of mouse were synthesized. After treating mouse neutrophils with vehicle (0.1 % DMSO) or 10 pM of mouse peptides 1 -9 for 30 minutes, flow cytometry was performed using propidium iodide (PI) and anti-Ly-6G antibodies. The viability was assessed in Ly-6G+neutrophils. ANOVA and Dunnett’s; **P < 0.01 , ***P < 0.001 , and ****p < 0.0001.

[0074] FIG. 4B is a graph showing Ca2+mobilization of neutrophils treated with peptide 1 , 2, or 3. WT neutrophils were pretreated with vehicle or 10 pM of each peptide. Ca2+mobilization was measured by Fura2 after stimulation with 5 pM fMLP, 0.5 pM A23187, or 10 pM Docket No.: 021025 / WO thapsigargin (TG) in the presence of 1 mM CaCL. ANOVA and Dunnett’s; *P < 0.05, **P < 0.01.

[0075] FIG. 4C is a graph showing Ca2+mobilization of neutrophils treated with peptide 4, 5, or 6. WT neutrophils were pretreated with vehicle or 10 pM of each peptide. Ca2+mobilization was measured by Fura2 after stimulation with 5 pM fMLP, 0.5 pM A23187, or 10 pM thapsigargin (TG) in the presence of 1 mM CaCh ANOVA and Dunnett’s; *P < 0.05.

[0076] FIG. 4D is a graph showing Ca2+mobilization of neutrophils treated with peptide 7, 8, or 9. WT neutrophils were pretreated with vehicle or 10 pM of each peptide. Ca2+mobilization was measured by Fura2 after stimulation with 5 pM fMLP, 0.5 pM A23187, or 10 pM thapsigargin (TG) in the presence of 1 mM CaCL. ANOVA and Dunnett’s; n.s.

[0077] FIG. 4E is a graph showing the surface level of aM[32 integrin binding in WT neutrophils were pretreated with vehicle or 10 pM of each peptide after treatment with 5 pM fMLP in the presence of 1 mM CaCL. ANOVA and Dunnett’s; *P < 0.05.

[0078] FIG. 4F is a graph showing the surface level of Alexa Fluor 488-conjugated fibrinogen (A488-FG) binding in WT neutrophils were pretreated with vehicle or 10 pM of each peptide after treatment with 5 pM fMLP in the presence of 1 mM CaCL. ANOVA and Dunnett’s; *P < 0.05.

[0079] FIG. 4G is an image of an immunoblot and corresponding graph. C57BL / 6 mouse neutrophils were pretreated with vehicle or 10 pM P2 or P3 and incubated with or without 5 pM fMLP for 2 minutes, followed by immunoprecipitation with anti-CRACR2A antibodies and immunoblotting with anti-STIM1 or anti-CRACR2A antibodies. Tukey’s test; *P < 0.05, **P < 0.01.

[0080] FIG. 4H is a set of representative images of C57BL / 6 mouse neutrophils. The neutrophils were pretreated with vehicle or 10 pM P2 or P3 for 30 minutes and stimulated with 5 pM fMLP. Cells were perfused on a monolayer of TNF-a-stimulated mouse pulmonary vein endothelial cells (ECs) for 5 minutes, followed by live imaging. The white arrowheads indicate rolling neutrophils, the grey arrowhead indicates crawling neutrophils, and the black arrowhead indicates TEM neutrophils. Bar = 50 pm.

[0081] FIG. 4I is a graph of the number of rolling neutrophils which were pretreated with vehicle or 10 pM P2 or P3 for 30 minutes and stimulated with 5 pM fMLP. Student’s t-test; *P < 0.05. Docket No.: 021025 / WO

[0082] FIG. 4J is a graph of the number of crawling neutrophils which were pretreated with vehicle or 10 pM P2 or P3 for 30 minutes and stimulated with 5 pM fMLP. Student’s t-test; ***P < 0.001.

[0083] FIG. 4K is a graph of the number of TEM) neutrophils which were pretreated with vehicle or 10 pM P2 or P3 for 30 minutes and stimulated with 5 pM fMLP. Student’s t-test; ***P < 0.001.

[0084] FIG. 4L is a schematic of the adoptive transfer experiment and IVM. I.V.: intravenous injection and I.P.: intraperitoneal injection.

[0085] FIG. 4M is a set of flow cytometry plots and corresponding graph showing the purity of Ly-6G+neutrophils was around 85%.

[0086] FIG. 4N is a set of immunofluorescent live tracking images of ear venules over time. An equal number of vehicle- or P2-treated neutrophils and a PE-conjugated anti-CD31 antibody were intravenously injected into recipient C57BL / 6 mice (n = 5) 2 hours after an intraperitoneal injection of TNF-a. One hour later, fluorescently labeled neutrophils were visualized on ear venules.

[0087] FIG. 40 is a graph showing the number of rolling neutrophils from vehicle- or P2- treated neutrophils intravenously injected into recipient C57BL / 6 mice (n = 5) as described in FIG. 4N. Mann-Whitney U test.

[0088] FIG. 4P is a graph showing the number of adherent neutrophils from vehicle- or P2- treated neutrophils intravenously injected into recipient C57BL / 6 mice (n = 5) as described in FIG. 4N. Mann-Whitney U test; ***P < 0.05.

[0089] FIG. 4Q is a graph showing the number of crawling neutrophils from vehicle- or P2- treated neutrophils intravenously injected into recipient C57BL / 6 mice (n = 5) as described in FIG. 4N. Mann-Whitney U test; ***P < 0.01.

[0090] FIG. 4R is a graph showing the viability of human neutrophils treated with peptides 1 - 9. Human neutrophils were pretreated with vehicle or 10 pM of human peptides 1 -9 (P1 -9) for 30 minutes. Flow cytometry was performed using PI and antibodies against CD66b, CD14, or CD15. The viability was assessed in CD66b+ / CD147CD15+neutrophils. ANOVA and Dunnett’s; **P < 0.01 , ***P < 0.001 , and ****P < 0.0001.

[0091] FIG. 4S is a graph showing Ca2+mobilization of human neutrophils treated with vehicle (0.1 % DMSO) or peptide 1 , 2, or 3 and after stimulation with 0.2 pM fMLP, 0.5 pM A23187, or 10 pM TG in the presence of 1 mM CaCE. ANOVA and Dunnett’s; *P < 0.05. Docket No.: 021025 / WO

[0092] FIG. 4T is a graph showing Ca2+mobilization of human neutrophils treated with vehicle (0.1 % DMSO) or peptide 4, 5, or 6 and after stimulation with 0.2 pM fMLP, 0.5 pM A23187, or 10 pM TG in the presence of 1 mM CaCl2. ANOVA and Dunnett’s.

[0093] FIG. 4U is a graph showing Ca2+mobilization of human neutrophils treated with vehicle (0.1 % DMSO) or peptide 7, 8, or 9 and after stimulation with 0.2 pM fMLP, 0.5 pM A23187, or 10 pM TG in the presence of 1 mM CaCh ANOVA and Dunnett’s; *P < 0.05.

[0094] FIG. 4V is a graph showing the surface level of total aM[32 in human neutrophils pretreated with vehicle or each peptide and then treated with 0.2 pM fMLP in the presence of 1 mM CaCl2 and 1 mM MgCL. The data represent the mean ± SD (n = 3-6). ANOVA and Dunnett’s; *P < 0.05, **P < 0.01.

[0095] FIG. 4W is a graph showing the surface level of activated aM[32 in human neutrophils pretreated with vehicle or each peptide and then treated with 0.2 pM fMLP in the presence of 1 mM CaCl2 and 1 mM MgCL. The data represent the mean ± SD (n = 3-6). ANOVA and Dunnett’s; *P < 0.05, ****P < 0.0001.

[0096] FIG. 4X is a graph showing the surface level of activated [32 in human neutrophils pretreated with vehicle or each peptide and then treated with 0.2 pM fMLP in the presence of 1 mM CaCl2 and 1 mM MgCL. The data represent the mean ± SD (n = 3-6). ANOVA and Dunnett’s; **P < 0.01 , ***P < 0.001 .

[0097] FIG. 5A is a set of representative images showing brain infarct in sham (left), WT (middle), and cracr2a CKO (right) mice. WT and cracr2a CKO mice were subjected to transient middle cerebral artery occlusion (tMCAO).

[0098] FIG. 5B is a graph measuring neurological behaviors as assessed by the Bederson score. ANOVA and Kruskal-Wallis test; **P < 0.01.

[0099] FIG. 5C is a graph measuring neurological behaviors as assessed by a grip strength test. ANOVA and Kruskal-Wallis test; *P < 0.05, ***P < 0.001 .

[0100] FIG. 5D is a graph of the infarct volume of WT and cracr2a CKO mice subjected to tMCAO. Infarct volume was measured by staining with 2,3,5-triphenyltetrazolium chloride. The bar indicates the median value in FIG. 5B and FIG. 5C, and the data represent the mean ± SD in D (n = 8 mice / group). Tukey’s test; **P < 0.01 , ****p < 0.0001.

[0101] FIG. 5E is a schematic of 4D intravital microscopy (IVM) and single-cell behavioral analysis. Four-dimensional confocal intravital imaging of pial and cortical microvessels was performed every 6 hours for 24 hours after tMCAO Docket No.: 021025 / WO

[0102] FIG. 5F is a set of representative single-plane images and a corresponding graph. The images show the maximal intensity projection (30 frames for 1 minute) of real-time rolling and crawling neutrophils in cerebral microvessels 24 hours after tM CAO. Bar = 100 pm; the white arrowheads indicate crawling neutrophils, and the yellow arrowheads indicate rolling neutrophils. The graph shows the intensity of aM[32 integrin was quantified in intravascular Ly-6G+neutrophils in WT and CKO mice. The bar indicates the median value (261 neutrophils for T and 173 neutrophils for CKO). Mann-Whitney U test; ****P < 0.0001 .

[0103] FIG. 5G is a graph showing the number of rolling neutrophils counted every 6 hours in WT and CKO mice after tMAAO and I V injections (FIG. 5E). The data represent the mean ± SD; two-way ANOVA and Sidak's multiple comparisons test.

[0104] FIG. 5H is a graph showing the number of crawling neutrophils counted every 6 hours in WT and CKO mice after tMAAO and I.V injections (FIG. 5E). The data represent the mean ± SD; two-way ANOVA and Sidak's multiple comparisons test; *P < 0.05.

[0105] FIG. 5I is a UMAP, doughnut, and density plots on WT and CKO neutrophils. Singlecell behavioral analysis of 434 intravascular neutrophils was performed for 10 minutes after combining behavioral data from WT and CKO mice 24 hours after tMCAO. The UMAP shows rolling and crawling behaviors in neutrophils. Doughnut and density plots show the distribution of three clusters of neutrophils and their different distribution in WT and CKO mice, respectively.

[0106] FIG. 5J is a set of violin plots for the track length (top), average speed (middle) and aM|32 intensity (bottom) of intravascular neutrophils in each cluster. The solid line and dotted line represent the median value and quartile, respectively (n = 5 mice / group). ANOVA and Kruskal-Wallis test; ****P < 0.0001 .

[0107] FIG. 5K is a set of violin plots for neutrophil aM[32 intensity in cluster 1 (left), cluster 2 (middle), and cluster 3 (right) between WT and CKO mice. The solid line and dotted line represent the median value and quartile, respectively (n = 5 mice / group). Mann-Whitney U test; *P < 0.05, ****P < 0.0001.

[0108] FIG. 5L is a set of representative 3D-rendered images and migration tracks of TEM neutrophils. Bar = 100 pm (upper images) and 30 pm (bottom images). Single-cell behavioral analysis of 313 transmigrated (TEM) neutrophils was performed for 10 minutes from WT and CKO mice 24 hours after tMCAO. Docket No.: 021025 / WO

[0109] FIG. 5M is a graph showing the number of TEM neutrophils from single-cell behavioral analysis of 313 transmigrated (TEM) neutrophils was performed for 10 minutes from WT and CKO mice 24 hours after tMCAO. The data represent the mean ± SD; two-way ANOVA and Sidak's multiple comparisons test; **P < 0.01.

[0110] FIG. 5N is a LIMAP, doughnut plot, and density plot of TEM neutrophils. The UMAP shows migratory behaviors in TEM neutrophils. Doughnut and density plots show the distribution of two subsets of TEM neutrophils and their different distribution in WT and CKO mice, respectively.

[0111] FIG. 50 is a set of violin plots for track length (top), average speed (middle), and aM[32 intensity (bottom) of TEM neutrophils in each cluster. The solid line and dotted line represent the median value and quartile, respectively (n = 5 mice / group). Mann-Whitney U test; ***P < 0.001 , ****P < 0.0001.

[0112] FIG. 5P is a set of violin plots for neutrophil aM[32 intensity in cluster 1 (left) and cluster 2 (right) in WT and CKO mice. The solid line and dotted line represent the median value and quartile, respectively (n = 5 mice / group). Mann-Whitney II test; *P < 0.05.

[0113] FIG. 5Q is a set of violin plots for neutrophil average speed in cluster 1 (left) and cluster 2 (right) in WT and CKO mice. The solid line and dotted line represent the median value and quartile, respectively (n = 5 mice / group). Mann-Whitney II test; *P < 0.05, ***P < 0.001.

[0114] FIG. 6A is a set of flow cytometry plots of WT and CKO Ly6G+ neutrophils. WT and cracr2a CKO mice were subjected to tMCAO and blood was obtained 24 hours after tMCAO to investigate neutrophil heterogeneity (n = 5-6 mice / group). The upper left quadrant represents aM[32highL-selectinl0Wneutrophils, and the lower right represents aM[32l0WL- selectinhighneutrophils.

[0115] FIG. 6B is a graph showing the surface level of L-selectin on peripheral neutrophils from WT and cracr2a CKO mice subjected to tMACO. Student’s t-test; *P < 0.05.

[0116] FIG. 6C is a graph showing the surface level of aM[32 integrin on peripheral neutrophils from WT and cracr2a CKO mice subjected to tMACO. Student’s t-test; *P < 0.05.

[0117] FIG. 6D is a graph showing the percentage of aM[32highL-selectinl0Wneutrophil populations. Student’s t-test; *P < 0.05.

[0118] FIG. 6E is a graph showing the percentage of aM[32l0WL-selectinhighneutrophil populations. Student’s t-test; **P < 0.01 . Docket No.: 021025 / WO

[0119] FIG. 6F is a set of flow cytometry plots of WT and CKO Ly6G+ neutrophils from mice subjected to tMCAO and blood was obtained 24 hours later. The upper right quadrant represents aMp2highfibrinogen(FG)highneutrophils.

[0120] FIG. 6G is a graph showing the surface level of Alexa Fluor 488-conjugated FG (A488- FG) on peripheral neutrophils from WT and CKO mice subjected to tMCAO. Student’s t-test; *P < 0.05.

[0121] FIG. 6H is a graph showing the percentage of aM[32highFGhighneutrophils in WT and CKO mice subjected to tMCAO. Student’s t-test; **P < 0.01 .

[0122] FIG. 6I is a set of flow cytometry plots of Ly-6G+ WT (left) and CKO (right) neutrophils labeled with aM[32 and L-selectin. Blood from WT and cracr2a CKO mice was collected every 4 hours for 24 hours. Neutrophils were labeled with Ly-6G, oM[32, and L-selectin, followed by flow cytometry (n = 5 mice / group). The upper left quadrant represents aM[32highL- selectinlow(aged) neutrophils, and the lower left quadrant represents aM[32l0WL-selectinhigh(young) neutrophils.

[0123] FIG. 6J is a plot of WT and cracr2a CKO neutrophils labeled with Ly-6G, aM|32, and L-selectin (n = 5 mice / group). The percentage of aM[32highL-selectinl0W(dotted line) or aMp2l0WL-selectinhigh(solid line) was plotted over time. The data represent the mean ± SD.

[0124] FIG. 7A is a schematic of the mouse cracr2a locus (top) and mRNA of cracr2a (bottom).

[0125] FIG. 7B is a set of images of RT-PCR of neutrophils from WT and cracr2a CKO mice. RNAs were isolated from WT and cracr2a CKO and probed for exon 3-4 (deletion site; top, left), exon 14-16 (long form; top, right), and Gapdh (control; bottom).

[0126] FIG. 8A is a graph of the speed of slowly rolling neutrophils on the ear venules. IVM data was analyzed to calculate the speed of slowly rolling neutrophils and the box graph indicates the 25thand 75thpercentiles. The middle line and whiskers of the box indicate the median and minimum / maximum values. **P < 0.01 versus WT control after Mann-Whitney U test.

[0127] FIG. 8B is a graph of the speed of slowly rolling neutrophils on the cremaster venules. IVM data was analyzed to calculate the speed of slowly rolling neutrophils and the box graph indicates the 25thand 75thpercentiles. The middle line and whiskers of the box indicate the median and minimum / maximum values. Mann-Whitney U test. Docket No.: 021025 / WO

[0128] FIG. 9A is a graph of rolling neutrophils from WT and cracr2a CKO mice on ear venules. The data represent the mean ± SD (n = 5). *P < 0.05.

[0129] FIG. 9B is a graph of adherent neutrophils from WT and cracr2a CKO mice on ear venules. The data represent the mean ± SD (n = 5). ****P < 0.0001 .

[0130] FIG. 9C is a graph of crawling neutrophils from WT and cracr2a CKO mice on ear venules. The data represent the mean ± SD (n = 5). *P < 0.05.

[0131] FIG. 9D is a graph of TEM neutrophils from WT and cracr2a CKO mice on ear venules. The data represent the mean ± SD (n = 5). ***P < 0.001 .

[0132] FIG. 9E is a graph of rolling neutrophils from WT and cracr2a CKO mice on cremaster venules. The data represent the mean ± SD (n = 5).

[0133] FIG. 9F is a graph of adherent neutrophils from WT and cracr2a CKO mice on cremaster venules. The data represent the mean ± SD (n = 5). *P < 0.05.

[0134] FIG. 9G is a graph of crawling neutrophils from WT and cracr2a CKO mice on cremaster venules. The data represent the mean ± SD (n = 5). *P < 0.05.

[0135] FIG. 9H is a graph of TEM neutrophils from WT and cracr2a CKO mice on cremaster venules. The data represent the mean ± SD (n = 5). ***P < 0.001.

[0136] FIG. 10A is a set of IVM images of WT (left) and cracr2a CKO (right) mice postcapillary venules. WT and cracr2a CKO mice were treated with an I.V injection of anti-CD42c, anti- CD31 , and anti-Ly-6G antibodies and an intraperitoneal injection of TNF-o, followed by intravital microscopy (IVM). White arrowheads indicate platelet-bound neutrophils. Bar = 50 pm.

[0137] FIG. 10B is a graph of the number of Pit-bound neutrophils from WT and cracr2a CKO mice treated with an I.V injection of anti-CD42c, anti-CD31 , and anti-Ly-6G antibodies and an intraperitoneal injection of TNF-a. Mann-Whitney U test; ****P < 0.0001.

[0138] FIG. 10C is a graph of the percentage of Pit-bound neutrophils from WT and cracr2a CKO mice treated with an I.V injection of anti-CD42c, anti-CD31 , and anti-Ly-6G antibodies and an intraperitoneal injection of TNF-a. Mann-Whitney U test; ****P < 0.0001.

[0139] FIG. 10D is a set of IVM images of WT (left) and cracr2a CKO (right) mice postcapillary venules. WT and cracr2a CKO mice were treated with an I.V. injection of anti-CD42c and anti-Ly-6G antibodies and an intrascrotal injection of TNF-a, followed by IVM. White arrowheads indicate platelet-bound neutrophils. Bar = 50 pm. Docket No.: 021025 / WO

[0140] FIG. 10E is a graph of the number of Pit-bound neutrophils from WT and cracr2a CKO mice treated with an I.V injection of anti-CD42c and anti-Ly-6G antibodies and an intrascrotal injection of TNF-a. Mann-Whitney U test; *P < 0.05.

[0141] FIG. 10F is a graph of the percentage of Pit-bound neutrophils from WT and cracr2a CKO mice treated with an I.V injection of anti-CD42c and anti-Ly-6G antibodies and an intrascrotal injection of TNF-a. Mann-Whitney U test; ****P < 0.0001.

[0142] FIG. 11 A is a graph of the number of neutrophils in WT and cracr2a CKO mice when treated with control IgG or R300 (anti-GPIba antibodies) for platelet depletion. The data represent the mean ± SD (n = 4); ANOVA and Tukey’s test.

[0143] FIG. 11 B is a graph of the number of platelets in WT and cracr2a CKO mice when treated with control IgG or R300 (anti-GPIba antibodies) for platelet depletion. The data represent the mean ± SD (n = 4); ANOVA and Tukey’s test; ****P < 0.0001 ).

[0144] FIG. 12A is a set of IVM images of neutrophil recruitment on ear venules from WT and cracr2a CKO mice treated with IgG or R300. One hour later, the mice were treated with an I.V. injection of anti-CD42c, anti-CD31 , and anti-Ly-6G antibodies and an I.P injection of TNF- a. IVM was performed 3 hours after TNF-a injection. The arrowheads (middle row) indicate transmigrated (TEM) neutrophils and the arrowheads (bottom row) indicate platelet-bound neutrophils. Bar = 50 pm; top row, merge; middle row, Ly6G; bottom row, CD42c.

[0145] FIG. 12B is a graph of the number of rolling neutrophils in WT and cracr2a CKO mice treated with IgG or R300, as described in FIG. 12A. The bar indicates the median value; ANOVA and Kruskal-Wallis; *P < 0.05, **P < 0.01 .

[0146] FIG. 12C is a graph of the number of adherent neutrophils in WT and cracr2a CKO mice treated with IgG or R300, as described in FIG. 12A. The bar indicates the median value; ANOVA and Kruskal-Wallis; *P < 0.05, **P < 0.01 .

[0147] FIG. 12D is a graph of the number of crawling neutrophils in WT and cracr2a CKO mice treated with IgG or R300, as described in FIG. 12A. The bar indicates the median value; ANOVA and Kruskal-Wallis; *P < 0.05, **P < 0.01 .

[0148] FIG. 12E is a graph of the number of TEM neutrophils in WT and cracr2a CKO mice treated with IgG or R300, as described in FIG. 12A. The bar indicates the median value; ANOVA and Kruskal-Wallis; *P < 0.05, **P < 0.01 . Docket No.: 021025 / WO

[0149] FIG. 12F is a graph of the number of rolling neutrophils in WT and cracr2a CKO mice treated with IgG or R300 analyzed by a linear mixed model. The bar indicates the median value; Tukey’s test; *P < 0.05, **P < 0.01.

[0150] FIG. 12G is a graph of the number of adherent neutrophils in WT and cracr2a CKO mice treated with IgG or R300 analyzed by a linear mixed model. The bar indicates the median value; Tukey’s test; *P < 0.05.

[0151] FIG. 12H is a graph of the number of crawling neutrophils in WT and cracr2a CKO mice treated with IgG or R300 analyzed by a linear mixed model. The bar indicates the median value; Tukey’s test; *P < 0.05.

[0152] FIG. 121 is a graph of the number of TEM neutrophils in WT and cracr2a CKO mice treated with IgG or R300 analyzed by a linear mixed model. The bar indicates the median value; Tukey’s test; *P < 0.05, **P < 0.01.

[0153] FIG. 13A is a set of representative IVM images of WT and cracr2a CKO neutrophils from mice treated with IgG or R300. WT and cracr2a CKO mice were pretreated with an intravenous injection of rat IgG or anti-GPIba antibodies (R300) as shown in Figure S5. One hour later, the mice were treated with an intravenous injection of anti-CD42c and anti-Ly-6G antibodies and an intrascrotal injection of TNF-a. Intravital microscopy (IVM) was performed 3 hours after TNF-a injection. The arrowheads indicate TEM neutrophils (middle row) and platelet-bound neutrophils (bottom row). Bar = 50 pm; top row, merge; middle row, Ly6G; bottom row, CD42c.

[0154] FIG. 13B is a graph of the number of rolling neutrophils in WT and cracr2a CKO mice treated with IgG or R300. The bar indicates the media value; ANOVA and Kruskal-Wallis test.

[0155] FIG. 13C is a graph of the number of adherent neutrophils in WT and cracr2a CKO mice treated with IgG or R300. The bar indicates the media value; ANOVA and Kruskal- Wallis test; *P < 0.05, **P < 0.01 .

[0156] FIG. 13D is a graph of the number of crawling neutrophils in WT and cracr2a CKO mice treated with IgG or R300. The bar indicates the media value; ANOVA and Kruskal- Wallis test; **P < 0.01.

[0157] FIG. 13E is a graph of the number of TEM neutrophils in WT and cracr2a CKO mice treated with IgG or R300. The bar indicates the media value; ANOVA and Kruskal-Wallis test; *P < 0.05, **P < 0.01 , ***P < 0.001 . Docket No.: 021025 / WO

[0158] FIG. 13F is a graph of the number of rolling neutrophils in WT and cracr2a CKO mice treated with IgG or R300 and analyzed in a linear mixed model. The bar indicates the mean ± SD (F-l, n = 10-11 venules in 3 mice / group); Tukey’s test.

[0159] FIG. 13G is a graph of the number of adherent neutrophils in WT and cracr2a CKO mice treated with IgG or R300 and analyzed in a linear mixed model. The bar indicates the mean ± SD (F-l, n = 10-11 venules in 3 mice / group); Tukey’s test; *P < 0.05, **P < 0.01.

[0160] FIG. 13H is a graph of the number of crawling neutrophils in WT and cracr2a CKO mice treated with IgG or R300 and analyzed in a linear mixed model. The bar indicates the mean ± SD (F-l, n = 10-11 venules in 3 mice / group); Tukey’s test; *P < 0.05, **P < 0.01.

[0161] FIG. 131 is a graph of the number of TEM neutrophils in WT and cracr2a CKO mice treated with IgG or R300 and analyzed in a linear mixed model. The bar indicates the mean ± SD (F-l, n = 10-11 venules in 3 mice / group); Tukey’s test; *P < 0.05.

[0162] FIG. 14A is a graph quantifying surface level L-selectin on neutrophils treated with or without 5 pM fMLP in the presence of 1 mM CaCh The data were obtained by the mean fluorescence intensity (MFI, mean ± SD, n = 4-5).

[0163] FIG. 14B is a graph quantifying surface level aL[32 on neutrophils treated with or without 5 pM fMLP in the presence of 1 mM CaCL. The data were obtained by the mean fluorescence intensity (MFI, mean ± SD, n = 4-5).

[0164] FIG. 14C is a graph quantifying surface level JAM-A on neutrophils treated with or without 5 pM fMLP in the presence of 1 mM CaCL. The data were obtained by the mean fluorescence intensity (MFI, mean ± SD, n = 4-5).

[0165] FIG. 14D is a graph quantifying surface level CD31 on neutrophils treated with or without 5 pM fMLP in the presence of 1 mM CaCL. The data were obtained by the mean fluorescence intensity (MFI, mean ± SD, n = 4-5).

[0166] FIG. 14E is a graph quantifying surface level PSGL-1 on neutrophils treated with or without 5 pM fMLP in the presence of 1 mM CaCL. The data were obtained by the mean fluorescence intensity (MFI, mean ± SD, n = 4-5).

[0167] FIG. 15 is a graph of gelatinase activity of WT and cracr2a-deficient neutrophils were treated with or without 5 pM fMLP or 0.5 pM A23187. The data represent the mean ± SD (n = 4). *p < 0.05 and **P < 0.01 versus WT control after Student’s f-test. Docket No.: 021025 / WO

[0168] FIG. 16A is a plot of cytosolic Ca2+release from WT and cracr2a-deficient neutrophils were labeled with FLIPR and after stimulation with or without 5 pM fMLP. Then, 1 mM CaCl2 was added to neutrophils, followed by measuring the influx.

[0169] FIG. 16B is a plot of cytosolic Ca2+release from WT and cracr2a-deficient neutrophils were labeled with FLIPR and after stimulation with or without 0.5 pM A23187. Then, 1 mM CaCL was added to neutrophils, followed by measuring the influx.

[0170] FIG. 16C is a plot of cytosolic Ca2+release from WT and cracr2a-deficient neutrophils were labeled with FLIPR and after stimulation with or without 10 pM TG. Then, 1 mM CaCL was added to neutrophils, followed by measuring the influx.

[0171] FIG. 16D is a graph quantifying Ca2+release by the area under the curve (AUC) from WT and cracr2a-deficient neutrophils were labeled with FLIPR and then treated with 5 pM fMLP, 0.5 pM A23187, or 10 pM TG. Student’s t-test; *P < 0.05, ***P < 0.001.

[0172] FIG. 16E is a graph quantifying Ca2+influx by the area under the curve (AUC) from WT and cracr2a-deficient neutrophils were labeled with FLIPR and then treated with 5 pM fMLP, 0.5 pM A23187, or 10 pM TG. Student’s t-test; *P < 0.05, **P < 0.01.

[0173] FIG. 16F is a graph quantifying the peak of Ca2+release from WT and cracr2a- deficient neutrophils were labeled with FLIPR and then treated with 5 pM fMLP, 0.5 pM A23187, or 10 pM TG. Student’s t-test; *P < 0.05, **P < 0.01 , ***P < 0.001.

[0174] FIG. 16G is a graph quantifying the peak of Ca2+influx from WT and cracr2a-deficient neutrophils were labeled with FLIPR and then treated with 5 pM fMLP, 0.5 pM A23187, or 10 pM TG. Student’s t-test; *P < 0.05, **P < 0.01 .

[0175] FIG. 17 is a graph of WT and cracr2a-deficient neutrophils labeled with calcein-AM, followed by flow cytometry. The data were obtained by the mean fluorescence intensity (MFI, mean ± SD, n = 3).

[0176] FIG. 18A is a plot of representative traces of reactive oxygen species (ROS) levels. WT and cracr2a-deficient neutrophils were incubated with 100 pM luminol and 8 U / ml horseradish peroxidase and then plated on fibrinogen-coated surfaces. After the addition of 20 pM fMLP to the cells, light emission was recorded for 60 minutes.

[0177] FIG. 18B is a plot of representative traces of reactive oxygen species (ROS) levels. WT and cracr2a-deficient neutrophils were incubated with 100 pM luminol and 8 U / ml horseradish peroxidase and then plated on fibrinogen-coated surfaces. After the addition of 50 nM PMA to the cells, light emission was recorded for 60 minutes. Docket No.: 021025 / WO

[0178] FIG. 18C is a graph quantifying the ROS production (FIG. 18A, FIG. 18B) by AUC. The data represent the mean ± SD (n = 5).

[0179] FIG. 19A is a table of two WT (E03 and E04) and two CRACR2A KO (E08 and F08) HL-60 cell lines were analyzed by next-generation sequencing (NGS) to confirm the deletion of CRACR2A.

[0180] FIG. 19B is a schematic of designed primers (sets 1 and 2) detecting around exon 6 of CRACR2A.

[0181] FIG. 19C is an image of a PCR gel for NGS set 1 (left) and NGS set 2 (right). The white arrowhead indicates exon 6-deleted products in the clone F08. WT (E04) and KO cells (F08) were used. Next-generation sequencing shows deletion of the target site in the exon 6 in HL-60 cells.

[0182] FIG. 20 is a graph of WT and CRACR2A KO dHL-60 cells treated with or without 5 pM fMLP or 0.25 pM A23187, followed by flow cytometry using anti-human PSGL-1 antibodies. The data represent the mean ± SD (n = 4). Deletion of CRACR2A does not alter the PSGL-1 level on the surface of dHL-60 cells.

[0183] FIG. 21 A is a schematic of the structure of the long (top) and short (bottom) forms of CRACR2A.

[0184] FIG. 21 B is a table of N-terminal palmitoylated eight 20-mers and one 16-mer of the coiled-coil region of mouse CRACR2A (amino acids 202-376).

[0185] FIG. 21 C is a table of N-terminal palmitoylated eight 20-mers and one 16-mer of the coiled-coil region of human CRACR2A (amino acids 207-382).

[0186] FIG. 22A is a graph of rolling neutrophils from C57BL / 6 mice were injected with anti- Ly-6G and anti-CD31 antibodies and either vehicle (1 .3% DMSO) or peptide 2 (P2, 13.3 pg / g mouse) via a tail vein into the mice right before or 2.5 hours after an intraperitoneal injection of TNF-a. IVM was performed 3 hours after TNF-a injection to visualize neutrophil on ear venules. The data represent the mean ± SD (n = 4 mice / group).

[0187] FIG. 22B is a graph of adherent neutrophils from C57BL / 6 mice were injected with anti-Ly-6G and anti-CD31 antibodies and either vehicle (1 .3% DMSO) or peptide 2 (P2, 13.3 pg / g mouse) via a tail vein into the mice right before or 2.5 hours after an intraperitoneal injection of TNF-a. IVM was performed 3 hours after TNF-a injection to visualize neutrophil on ear venules. The data represent the mean ± SD (n = 4 mice / group). Docket No.: 021025 / WO

[0188] FIG. 22C is a graph of crawling neutrophils from C57BL / 6 mice were injected with anti- Ly-6G and anti-CD31 antibodies and either vehicle (1 .3% DMSO) or peptide 2 (P2, 13.3 pg / g mouse) via a tail vein into the mice right before or 2.5 hours after an intraperitoneal injection of TNF-a. IVM was performed 3 hours after TNF-a injection to visualize neutrophil on ear venules. The data represent the mean ± SD (n = 4 mice / group).

[0189] FIG. 22D is a graph of TEM neutrophils from C57BL / 6 mice were injected with anti- Ly-6G and anti-CD31 antibodies and either vehicle (1 .3% DMSO) or peptide 2 (P2, 13.3 pg / g mouse) via a tail vein into the mice right before or 2.5 hours after an intraperitoneal injection of TNF-a. IVM was performed 3 hours after TNF-a injection to visualize neutrophil on ear venules. The data represent the mean ± SD (n = 4 mice / group).

[0190] FIG. 23A is a schematic of human peripheral T lymphocytes, monocytes, and neutrophils isolated by a discontinuous Percoll density gradient.

[0191] FIG. 23B is a set of flow cytometry plots showing the purity of human peripheral T lymphocytes, monocytes, and neutrophils isolated from human blood as shown in FIG. 23A.

[0192] FIG. 24A is a violin plot of the displacement length of neutrophils in each cluster. Single-cell behavioral analysis of 434 intravascular neutrophils was performed on data from WT and cracr2a CKO mice 24 hours after tMCAO. The data represent the median (solid line) and quartiles (dotted line). ****P < 0.0001 ; ANOVA and Kruskal-Wallis test with post hoc Dunn correction.

[0193] FIG. 24B is a violin plot of the straightness of neutrophils in each cluster. Single-cell behavioral analysis of 434 intravascular neutrophils was performed on data from WT and cracr2a CKO mice 24 hours after tMCAO. The data represent the median (solid line) and quartiles (dotted line). **P < 0.01 ****P < 0.0001 ; ANOVA and Kruskal-Wallis test with post hoc Dunn correction.

[0194] FIG. 24C is a violin plot of the duration of neutrophils in each cluster. Single-cell behavioral analysis of 434 intravascular neutrophils was performed on data from WT and cracr2a CKO mice 24 hours after tMCAO. The data represent the median (solid line) and quartiles (dotted line). **P < 0.01 ****P < 0.0001 ; ANOVA and Kruskal-Wallis test with post hoc Dunn correction.

[0195] FIG. 25A is a set of representative immunofluorescent images of WT and cracr2a CKO mice. Platelet-neutrophil interaction is not detected on pial and cortex microvessels of mice following focal brain ischemia. WT and cracr2a CKO mice were subjected to tMCAO and Docket No.: 021025 / WO cerebral intravital microscopy was performed 18 and 24 hours after tMCAO. arrowheads indicate aM|32highadherent or crawling neutrophils. Bar = 50 pm.

[0196] FIG. 25B is a graph of the number of platelets-bound aM[32highneutrophils. The graph indicates the median value (n = 7 vessels in 3 mice / group).

[0197] FIG. 26A is a schematic of CRACR2a-L protein and the various domains.

[0198] FIG. 26B is a schematic of palmitoylated peptide to bind to CRACR2A.

[0199] FIG. 27A is a graph of L-selectin on neutrophils incubated with the indicated peptide and treated with control, 10 pM fMLP, or 20ng / ml TNF-a.

[0200] FIG. 27B is a graph of aM[32 integrin on neutrophils incubated with the indicated peptide and treated with control, 10 pM fMLP, or 20ng / ml TNF-a.

[0201] FIG. 27C is a plot and corresponding graph of cytosolic Ca2+from neutrophils treated with peptide 5 or peptide 6 (FIG. 26B) and stimulation with or without 5 pM fMLP. The graph measures the AUC of cytosolic Ca2+.

[0202] FIG. 28A is a graph of the number of rolling neutrophils when treated with peptide 5 and peptide 6.

[0203] FIG. 28B is a graph of the number of adherent neutrophils when treated with peptide 5 and peptide 6.

[0204] FIG. 28C is a graph of the number of crawling neutrophils when treated with peptide 5 and peptide 6.

[0205] FIG. 28D is a graph of the number of TEM neutrophils when treated with peptide 5 and peptide 6.

[0206] FIG. 29A is a plot of cytosolic Ca2+from WT and CKO neutrophils treated with or without Ca2+.

[0207] FIG. 29B is a plot of cytosolic Ca2+from WT and CKO neutrophils treated with or without TNF-a.

[0208] FIG. 29C is a graph measuring the AUC of cytosolic Ca2+from WT and CKO neutrophils which are unstimulated or treated with Ca2+or TNF-a.

[0209] FIG. 29D is a graph measuring the AUC of cytosolic Ca2+from WT and CKO neutrophils which are unstimulated or treated with fMLP, A23187, orTG.

[0210] DETAILED DESCRIPTION OF THE DISCLOSURE

[0211] The present disclosure is based, at least in part, on the discovery that deletion of Docket No.: 021025 / WO neutrophil CRACR2A significantly reduces neutrophil adhesive and migratory function under inflammatory conditions but does not alter ROS production. Accordingly, the present disclosure demonstrates CRACR2A as a therapeutic target for neutrophil-mediated inflammatory diseases and targeting CRACR2A may not impair innate immune responses. A palmitoylated 20-mer (AAYDEEIQHLYEEMEQQIKS (SEQ ID NO: 17)) was found to effectively block CRACR2A-STIM1 interaction and reduces cytosolic Ca2+ level, beta2 integrin activation, and neutrophil adhesive function under inflammatory conditions.

[0212] A clinical study and GWAS search reveal that Ca2+ release-activated Ca2+ channel regulator 2A (CRACR2A) is linked to immunodeficiency due to T-cell dysfunction and inflammatory diseases in humans. However, prior to the present disclosure, the role of neutrophil CRACR2A in acute inflammation was unknown.

[0213] Using real-time 4D intravital microscopy with myeloid-specific cracr2a conditional knockout mice, it was discovered herein that deletion of neutrophil CRACR2A significantly reduces neutrophil recruitment on peripheral blood vessels in mouse models of TNF-alpha- induced systemic and local inflammation. Furthermore, it was found that compared to WT control mice, cracr2a CKO mice exhibit a significant reduction in brain damage in mice subjected to transient middle cerebral artery occlusion (a mouse model of ischemic stroke).

[0214] Mechanistically, CRACR2A interacts with stromal interaction molecule 1 (STIM1 ) and increases cytosolic Ca2+ levels, thereby promoting beta2 integrin activation and facilitating neutrophil infiltration into inflamed tissues. Due to the lack of the pharmacologic inhibitors of CRACR2A, eight 20-mers (P1 -P8) and one 16-mer (P9) were designed and synthesized covering the STIM1 -binding region in CRACR2A. In order for the peptides to get into the cells, nine peptides were palmitoylated at the N-terminus. It was found that palmitoylated P2 (AAYDEEIQHLYEEMEQQIKS (SEQ ID NO: 17), amino acids 221-240 in mouse cracr2a and amino acids 227-246 in human CRACR2A), is identical in mouse and human CRACR2A and inhibits the STIM1-CRACR2A interaction. When the peptide was tested in various in vitro and ex vivo studies, treatment of neutrophils with 10 microM palmitoylated P2 reduces cytosolic Ca2+ levels and beta2 integrin function in stimulated neutrophils and blocks neutrophil adhesion and transmigration on a monolayer of TNF-alphastimulated mouse vein endothelial cells under venous blood shear conditions. These results provide strong evidence that blocking CRACR2A-STIM1 interaction with palmitoylated P2 may be a therapeutic potential to mitigate neutrophil-mediated inflammatory diseases. Docket No.: 021025 / WO

[0215] ANTI-INFLAMMATORY PEPTIDES

[0216] Anti-inflammatory peptides of the present disclosure include CRACR2A-derived peptides, such as peptides derived from the stromal interaction molecule 1 (STIMI )-binding region in CRACR2A. In exemplary embodiments, at least one anti-inflammatory peptide is included in compositions for administration to a subject in need of STIM1 -CRACR2A inhibition, such as for reducing inflammation, treating ischemic stroke, preventing inflammation-induced tissue injury, and other methods of use thereof as described herein. In some embodiments, the peptides are palmitoylated and / or N-terminal as described herein. Depending on the embodiment, an anti-inflammatory peptide of the present disclosure is not less than a 10-mer peptide and is not more than a 50-mer peptide. In some embodiments an anti-inflammatory peptide is a 16-mer peptide, such as (but not limited to) P9. In some embodiments, an anti-inflammatory peptide is a 20-mer peptide, such as (but not limited to) P1 -P8. In some embodiments, an anti-inflammatory peptide can be selected from Table 1 and Table 2.

[0217] Compositions of the present disclosure include at least one anti-inflammatory peptide. Anti-inflammatory peptides of the exemplary embodiments include isolated and / or synthesized peptides derived from (covering) a coiled-coil region of CRACR2A. In some exemplary embodiments the at least one anti-inflammatory peptide is palmitoylated.

[0218] Table 1 : N-terminal palmitoylated mers of the coiled-coil region of mouse CRACR2A (amino acids 202-376).

[0219] Table 2: N-terminal palmitoylated mers of the coiled-coil region of human CRACR2A (amino acids 207-382). Docket No.: 021025 / WO

[0220] MODULATION AGENTS

[0221] As described herein, gene and / or associated protein expression has been implicated in various diseases, disorders, and conditions. As such, modulation of gene and protein expression can be used for treatment of such conditions. A modulation agent can modulate response, such as by inducing or inhibiting gene and / or protein expression signaling. Modulation can comprise modulating protein expression on cells, modulating the quantity of gene / protein expressing cells, or modulating the quality of gene / protein expressing cells.

[0222] Modulation agents can be any composition or method that modulates expression on cells. For example, a modulation agent can be an activator, an inhibitor, an agonist, or an antagonist. As another example, the modulation can be the result of gene editing.

[0223] A modulation agent can be an antibody (e.g., a monoclonal antibody). A modulating agent can be an agent that induces or inhibits progenitor cell differentiation into gene / protein expressing cells.

[0224] SIGNAL REDUCTION, ELIMINATION, OR INHIBITION BY SMALL MOLECULE INHIBITORS, SHRNA, SIRNA, ORASOS

[0225] As described herein, a modulation and / or inhibiting agent can be used for use in various therapies. A modulation agent can be used to reduce / eliminate or enhance / increase cellular pathway signals. For example, a modulation agent can be a small molecule inhibitor. As another example, a modulation agent can be a short hairpin RNA (shRNA). As another example, a modulation agent can be a short interfering RNA (siRNA).

[0226] As another example, RNA (e.g., long noncoding RNA (IncRNA)) can be targeted with antisense oligonucleotides (ASOs) as a therapeutic. Processes for making ASOs targeted to Docket No.: 021025 / WO

[0227] RNAs are well known; see e.g., Zhou et al. 2016 Methods Mol Biol. 1402:199-213. Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0228] INHIBITING AGENT

[0229] One aspect of the present disclosure provides for targeting of CRACR2A, its receptor, or its downstream signaling. The present disclosure provides methods of treating or preventing inflammation, inflammation-induced tissue injury, and / or ischemic stroke based on the discovery that CRACR2A derived anti-inflammatory peptides were found to effectively block CRACR2A-STIM1 interaction and reduce: cytosolic Ca2+level, beta2 integrin activation, and neutrophil adhesive function under inflammatory conditions.

[0230] As described herein, inhibitors or antagonists (e.g., antibodies, fusion proteins, small molecules) can reduce or prevent signaling, binding, activation and / or function. An inhibiting agent can be any agent that can inhibit activity, inhibit signaling, downregulate protein level, downregulate expression, or knockdown gene expression.

[0231] For example, the inhibiting agent can be an anti-CRACR2A antibody or an anti-STIM1 / CRACR2A antibody, wherein the anti-type antibody prevents binding to a receptor, or prevents activation of downstream signaling. Furthermore, the antibody can be a murine antibody, a humanized murine antibody, or a human antibody.

[0232] As another example, the inhibiting agent can be a fusion protein. For example, the fusion protein can be a decoy receptor. Furthermore, the fusion protein can comprise a mouse or human Fc antibody domain fused to an ectodomain.

[0233] The CRACR2A-inhibiting agent(s) of the present disclosure can be anti-inflammatory peptides covering the coiled-coil region of CRACR2A (e.g., peptides derived from the stromal interaction molecule 1 (STIMI )-binding region in CRACR2A, which are demonstrated herein to be a potent and specific inhibitor of STIM1-CRACR2A interaction / signaling.

[0234] For example, an inhibiting agent can be an inhibitory protein such as an antagonist. As disclosed herein, a CRACR2A-inhibiting agent can be an inhibitory protein (or peptide) that antagonizes / blocks an STIM1 -CRACR2A interaction. As another example, the inhibiting agent can be a viral protein antagonist. As another example, an inhibiting agent can be a short hairpin RNA (shRNA) or a short interfering RNA (siRNA). As another example, an inhibiting agent can be a single guide RNA (sgRNA).

[0235] Methods for preparing an inhibiting agent, such as the CRACR2A-inhibiting agents Docket No.: 021025 / WO described herein (e.g., an agent capable of inhibiting STIM1 -CRACR2A interactions and / or pathway signaling) can comprise construction of a protein / Ab scaffold containing the natural receptor as a neutralizing agent; developing inhibitors of a receptor “down-stream”; or developing inhibitors of production “up-stream”.

[0236] Inhibiting can be performed by genetically modification in a subject or genetically modifying a subject to reduce or prevent expression of a target gene, such as through the use of CRISPR-Cas9 or analogous technologies.

[0237] Inhibition of agents as described herein can be determined by standard pharmaceutical procedures in assays or cell cultures for determining the ICso. The half maximal inhibitory concentration (ICso) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. The ICso is a quantitative measure that indicates how much of a particular inhibitory substance (e.g., pharmaceutical agent or drug) is needed to inhibit, in vitro, a given biological process or biological component by 50%. The biological component could be an enzyme, cell, cell receptor, or microorganism, for example. ICso values are typically expressed as molar concentration. ICso is generally used as a measure of antagonist drug potency in pharmacological research. ICso is comparable to other measures of potency, such as ECso for excitatory drugs. ECso represents the dose or plasma concentration required for obtaining 50% of a maximum effect in vivo. ICso can be determined with functional assays or with competition binding assays.

[0238] CHEMICAL AGENT

[0239] Examples of CRACR2A-inhibiting agents (e.g., STIM1 -CRACR2A interaction inhibiting agents) are described herein. Agents can include one or more anti-inflammatory peptides as disclosed herein, or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, prodrug, analog, or stereoisomer thereof or optionally substituted analog thereof.

[0240] The formulas, analogs, and R groups can be optionally substituted or functionalized with one or more groups independently selected from the group consisting of hydroxyl; Ci- walkyl hydroxyl; amine; Ci-wcarboxylic acid; Ci-wcarboxyl; straight chain or branched Ci- walkyl, optionally containing unsaturation; a C2- cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched Ci-walkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, 0, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein the unsubstituted phenyl ring or substituted phenyl ring can Docket No.: 021025 / WO be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; Ci- alkyl hydroxyl; amine; Ci- carboxyl; Ci- carboxylic acid; Ci- wcarboxyl; straight chain or branched Ci-walkyl, optionally containing unsaturation; straight chain or branched Ci-walkyl amine, optionally containing unsaturation; a C2-iocycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched Ci-walkyl amine; heterocyclyl; heterocyclic amine; aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, 0, or S atoms; and the unsubstituted heterocyclyl or substituted heterocyclyl can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; Ci-walkyl hydroxyl; amine; Ci-wcarboxylic acid; Ci- wcarboxyl; straight chain or branched Ci-walkyl, optionally containing unsaturation; straight chain or branched Ci-walkyl amine, optionally containing unsaturation; a C2-iocycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; heterocyclyl; straight chain or branched Ci-walkyl amine; heterocyclic amine; and aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, 0, or S atoms. Any of the above can be further optionally substituted.

[0241] The term “imine” or “imino”, as used herein, unless otherwise indicated, can include a functional group or chemical compound containing a carbon-nitrogen double bond. The expression “imino compound”, as used herein, unless otherwise indicated, refers to a compound that includes an “imine” or an “imino” group as defined herein. The “imine” or “imino” group can be optionally substituted.

[0242] The term “hydroxyl”, as used herein, unless otherwise indicated, can include -OH. The “hydroxyl” can be optionally substituted.

[0243] The terms “halogen” and “halo”, as used herein, unless otherwise indicated, include a chlorine, chloro, Cl; fluorine, fluoro, F; bromine, bromo, Br; or iodine, iodo, or I.

[0244] The term “acetamide”, as used herein, is an organic compound with the formula CH3CONH2. The “acetamide” can be optionally substituted.

[0245] The term “aryl”, as used herein, unless otherwise indicated, include a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, or anthracenyl. The “aryl” can be optionally substituted.

[0246] The terms “amine” and “amino”, as used herein, unless otherwise indicated, include a functional group that contains a nitrogen atom with a lone pair of electrons and wherein one or more hydrogen atoms have been replaced by a substituent such as, but not limited to, an Docket No.: 021025 / WO alkyl group or an aryl group. The “amine” or “amino” group can be optionally substituted.

[0247] The term “alkyl”, as used herein, unless otherwise indicated, can include saturated monovalent hydrocarbon radicals having straight or branched moieties, such as but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl groups, etc. Representative straightchain lower alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n- pentyl, -n-hexyl, -n-heptyl and -n-octyl; while branched lower alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 2- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3- dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, unsaturated Ci -10 alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1 -pentenyl, -2-pentenyl, -3-methyl-1 -butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1 -hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl, -2- butynyl, -1 -pentynyl, -2-pentynyl, or -3-methyl-1 butynyl. An alkyl can be saturated, partially saturated, or unsaturated. The “alkyl” can be optionally substituted.

[0248] The term “carboxyl”, as used herein, unless otherwise indicated, can include a functional group consisting of a carbon atom double bonded to an oxygen atom and single bonded to a hydroxyl group (-COOH). The “carboxyl” can be optionally substituted.

[0249] The term “carbonyl”, as used herein, unless otherwise indicated, can include a functional group consisting of a carbon atom double-bonded to an oxygen atom (C=O). The “carbonyl” can be optionally substituted.

[0250] The term “alkenyl”, as used herein, unless otherwise indicated, can include alkyl moieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety. An alkenyl can be partially saturated or unsaturated. The “alkenyl” can be optionally substituted.

[0251] The term “alkynyl”, as used herein, unless otherwise indicated, can include alkyl moieties having at least one carbon-carbon triple bond wherein alkyl is as defined above. An alkynyl can be partially saturated or unsaturated. The “alkynyl” can be optionally substituted.

[0252] The term “acyl”, as used herein, unless otherwise indicated, can include a functional group derived from an aliphatic carboxylic acid, by removal of the hydroxyl (-OH) group. The “acyl” can be optionally substituted.

[0253] The term “alkoxyl”, as used herein, unless otherwise indicated, can include O-alkyl Docket No.: 021025 / WO groups wherein alkyl is as defined above and 0 represents oxygen. Representative alkoxyl groups include, but are not limited to, -O-methyl, -O-ethyl, -O-n-propyl, -O-n-butyl, -O-n- pentyl, -O-n-hexyl, -O-n-heptyl, -O-n-octyl, -O-isopropyl, -O-sec-butyl, -O-isobutyl, -O-tert- butyl, -O-isopentyl, -O-2-methylbutyl, -O-2-methylpentyl, -O-3-methylpentyl, -0-2,2- dimethylbutyl, -0-2,3-dimethylbutyl, -0-2,2-dimethylpentyl, -0-2,3-dimethylpentyl, -0-3,3- dimethylpentyl, -0-2,3,4-trimethylpentyl, -0-3-methylhexyl, -0-2,2-dimethylhexyl, -0-2,4- dimethylhexyl, -0-2,5-dimethylhexyl, -0-3,5-dimethylhexyl, -O-2,4dimethylpentyl, -0-2- methylheptyl, -0-3-methylheptyl, -O-vinyl, -O-allyl, -0-1-butenyl, -0-2-butenyl, -0- isobutylenyl, -0-1 -pentenyl, -0-2-pentenyl, -0-3-methyl-1 -butenyl, -O-2-methyl-2-butenyl, - O-2,3-dimethyl-2-butenyl, -0-1 -hexyl, -0-2-hexyl, -0-3-hexyl, -O-acetylenyl, -O-propynyl, - 0-1-butynyl, -0-2-butynyl, -0-1 -pentynyl, -0-2-pentynyl and -0-3-methyl-1-butynyl, -0- cyclopropyl, -O-cyclobutyl, -O-cyclopentyl, -O-cyclohexyl, -O-cycloheptyl, -O-cyclooctyl, -0- cyclononyl and -O-cyclodecyl, -O-CH2-cyclopropyl, -O-CH2-cyclobutyl, -O-CH2-cyclopentyl, - O-CH2-cyclohexyl, -O-CH2-cycloheptyl, -O-CH2-cyclooctyl, -0- CH2-cyclononyl, -O-CH2- cyclodecyl, -O-(CH2)2-cyclopropyl, -O-(CH2)2-cyclobutyl, -O-(CH2)2-cyclopentyl, -O-(CH2)2- cyclohexyl, -O-(CH2)2-cycloheptyl, -O-(CH2)2-cyclooctyl, -O-(CH2)2-cyclononyl, or -O-(CH2)2- cyclodecyl. An alkoxyl can be saturated, partially saturated, or unsaturated. The “alkoxyl” can be optionally substituted.

[0254] The term “cycloalkyl”, as used herein, unless otherwise indicated, can include an aromatic, a non-aromatic, saturated, partially saturated, or unsaturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 1 to 10 carbon atoms (e.g., 1 or 2 carbon atoms if there are other heteroatoms in the ring), preferably 3 to 8 ring carbon atoms. Examples of cycloalkyls include, but are not limited to, C3-10 cycloalkyl groups include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1 ,3-cyclohexadienyl, -1 ,4-cyclohexadienyl, - cycloheptyl, -1 ,3-cycloheptadienyl, -1 ,3,5-cycloheptatrienyl, -cyclooctyl, and cyclooctadienyl. The term “cycloalkyl” also can include -lower alky l-cycloalkyl, wherein lower alkyl and cycloalkyl are as defined herein. Examples of -lower alky l-cycloalkyl groups include, but are not limited to, -CH2-cyclopropyl, -CH2-cyclobutyl, -CH2-cyclopentyl, -CH2- cyclopentadienyl, -CH2-cyclohexyl, -CH2-cycloheptyl, or -CH2-cyclooctyl. The “cycloalkyl” can be optionally substituted. A “cycloheteroalkyl”, as used herein, unless otherwise indicated, can include any of the above with a carbon substituted with a heteroatom (e.g., O, S, N). Docket No.: 021025 / WO

[0255] The term “heterocyclic” or “heteroaryl”, as used herein, unless otherwise indicated, can include an aromatic or non-aromatic cycloalkyl in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of 0, S, and N. Representative examples of a heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1 ,4)-dioxane, (1 ,3)-dioxolane, 4,5- dihydro-1 H-imidazolyl, or tetrazolyl. Heterocycles can be substituted or unsubstituted. Heterocycles can also be bonded at any ring atom (i.e., at any carbon atom or heteroatom of the heterocyclic ring). A heterocyclic can be saturated, partially saturated, or unsaturated. The “heterocyclic” can be optionally substituted.

[0256] The term “indole”, as used herein, is an aromatic heterocyclic organic compound with formula C8H7N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. The “indole” can be optionally substituted.

[0257] The term “cyano”, as used herein, unless otherwise indicated, can include a -CN group. The “cyano” can be optionally substituted.

[0258] The term “alcohol”, as used herein, unless otherwise indicated, can include a compound in which the hydroxyl functional group (-0H) is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms. The “alcohol” can be optionally substituted.

[0259] The term “solvate” is intended to mean a solvate form of a specified compound that retains the effectiveness of such compound. Examples of solvates include compounds of the invention in combination with, for example, water, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.

[0260] The term “mmol”, as used herein, is intended to mean millimole. The term “equiv”, as used herein, is intended to mean equivalent. The term “mL”, as used herein, is intended to mean milliliter. The term “g”, as used herein, is intended to mean gram. The term “kg”, as used herein, is intended to mean kilogram. The term “pg”, as used herein, is intended to mean micrograms. The term “h”, as used herein, is intended to mean hour. The term “min”, as used herein, is intended to mean minute. The term “M”, as used herein, is intended to mean molar. The term "pL", as used herein, is intended to mean microliter. The term “pM”, Docket No.: 021025 / WO as used herein, is intended to mean micromolar. The term “nM”, as used herein, is intended to mean nanomolar. The term “N”, as used herein, is intended to mean normal. The term “amu”, as used herein, is intended to mean atomic mass unit. The term “°C”, as used herein, is intended to mean degree Celsius. The term “wt / wt”, as used herein, is intended to mean weight / weight. The term “v / v”, as used herein, is intended to mean volume / volume. The term “MS”, as used herein, is intended to mean mass spectroscopy. The term “HPLC”, as used herein, is intended to mean high performance liquid chromatograph. The term “RT”, as used herein, is intended to mean room temperature. The term "e.g.", as used herein, is intended to mean example. The term “N / A”, as used herein, is intended to mean not tested.

[0261] As used herein, the expression “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, or pamoate (i.e., 1 ,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion, or another counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In instances where multiple charged atoms are part of the pharmaceutically acceptable salt, the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion. As used herein, the expression “pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. As used herein, the expression “pharmaceutically acceptable hydrate” refers to a compound of the invention, or a salt thereof, that further can include a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. Docket No.: 021025 / WO

[0262] MOLECULAR ENGINEERING

[0263] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0264] The term “transfection,” as used herein, refers to the process of introducing nucleic acids into cells by non-viral methods. The term “transduction,” as used herein, refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

[0265] The terms "heterologous DNA sequence", "exogenous DNA segment", or "heterologous nucleic acid”, “transgene”, “exogenous polynucleotide” as used herein, each refers to a sequence that originates from a source foreign (e.g., non-native) to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0266] Sequences described herein can also be the reverse, the complement, or the reverse complement of the nucleotide sequences described herein. The RNA goes in the reverse direction compared to the DNA, but its base pairs still match (e.g., G to C). The reverse complementary RNA for a positive strand DNA sequence will be identical to the corresponding negative strand DNA sequence. Reverse complement converts a DNA sequence into its reverse, complement, or reverse-complement counterpart. Docket No.: 021025 / WO

[0267] Complementarity is a property shared between two nucleic acid sequences (e.g., RNA, DNA), such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary. Two bases are complementary if they form Watson- Crick base pairs.

[0268] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0269] An “expression vector”, otherwise known as an “expression construct”, is generally a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and / or promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively. As described herein, Escherichia coli is used as the host for Docket No.: 021025 / WO protein production, but other cell types may also be used.

[0270] In molecular biology, an “inducer” is a molecule that regulates gene expression. An inducer can function in two ways, such as:

[0271] (i) By disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator. RNA polymerase can then begin to transcribe operon genes. An operon is a cluster of genes that are transcribed together to give a single messenger RNA (mRNA) molecule, which therefore encodes multiple proteins.

[0272] (ii) By binding to activators. Activators generally bind poorly to activator DNA sequences unless an inducer is present. An activator binds to an inducer and the complex binds to the activation sequence and activates target gene. Removing the inducer stops transcription. Because a small inducer molecule is required, the increased expression of the target gene is called induction.

[0273] Repressor proteins bind to the DNA strand and prevent RNA polymerase from being able to attach to the DNA and synthesize mRNA. Inducers bind to repressors, causing them to change shape and preventing them from binding to DNA. Therefore, they allow transcription, and thus gene expression, to take place.

[0274] For a gene to be expressed, its DNA sequence (or polynucleotide sequence) must be copied (in a process known as transcription) to make a smaller, mobile molecule called messenger RNA (mRNA), which carries the instructions for making a protein to the site where the protein is manufactured (in a process known as translation). Many different types of proteins can affect the level of gene expression by promoting or preventing transcription. In prokaryotes (such as bacteria), these proteins often act on a portion of DNA known as the operator at the beginning of the gene. The promoter is where RNA polymerase, the enzyme that copies the genetic sequence and synthesizes the mRNA, attaches to the DNA strand.

[0275] Some genes are modulated by activators, which have the opposite effect on gene expression as repressors. Inducers can also bind to activator proteins, allowing them to bind to the operator DNA where they promote RNA transcription. Ligands that bind to deactivate activator proteins are not, in the technical sense, classified as inducers, since they have the effect of preventing transcription.

[0276] A “promoter” is generally understood as a nucleic acid control sequence that directs Docket No.: 021025 / WO transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0277] A “ribosome binding site”, or “ribosomal binding site (RBS)”, refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of translation. Generally, RBS refers to bacterial sequences, although internal ribosome entry sites (IRES) have been described in mRNAs of eukaryotic cells or viruses that infect eukaryotes. Ribosome recruitment in eukaryotes is generally mediated by the 5' cap present on eukaryotic mRNAs.

[0278] A ribosomal skipping sequence (e.g., 2A sequence such as furin-GSG-T2A) can be used in a construct to prevent covalently linking translated amino acid sequences.

[0279] A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN- 10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

[0280] The “transcription start site” or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1 . With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' Docket No.: 021025 / WO direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0281] "Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0282] A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

[0283] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0284] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

[0285] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism such Docket No.: 021025 / WO as a bacterium, cyanobacterium, animal, or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using self-replicating primers, paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransformed" refers to normal cells that have not been through the transformation process.

[0286] "Wild-type" refers to a virus or organism found in nature without any known mutation.

[0287] Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991 ) Gene 97(1 ), 119-123; Ghadessy et al. (2001 ) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0288] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those 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. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X / Y100, where X is the number of Docket No.: 021025 / WO residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. For example, the percent identity can be at least 80% or about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0289] Substitution refers to the replacement of one amino acid with another amino acid in a protein or the replacement of one nucleotide with another in DNA or RNA. Insertion refers to the insertion of one or more amino acids in a protein or the insertion of one or more nucleotides with another in DNA or RNA. Deletion refers to the deletion of one or more amino acids in a protein or the deletion of one or more nucleotides with another in DNA or RNA. Generally, substitutions, insertions, or deletions can be made at any position so long as the required activity is retained.

[0290] “Point mutation” refers to when a single base pair is altered. A point mutation or substitution is a genetic mutation where a single nucleotide base is changed, inserted, or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product — consequences that are moderately predictable based upon the specifics of the mutation. These consequences can range from no effect (e.g., synonymous mutations) to deleterious effects (e.g., frameshift mutations), with regard to protein production, composition, and function. Point mutations can have one of three effects. First, the base substitution can be a silent mutation where the altered codon corresponds to the same amino acid. Second, the base substitution can be a missense mutation where the altered codon corresponds to a different amino acid. Or third, the base substitution can be a nonsense mutation where the altered codon corresponds to a stop signal. Silent mutations result in a new codon (a triplet nucleotide sequence in RNA) that codes for the same amino acid as the wild type codon in that position. In some silent mutations the codon codes for a different amino acid that happens to have the same properties as the amino acid produced by the wild type codon. Missense mutations involve substitutions that result in functionally different amino acids; these can lead to alteration or loss of protein function. Nonsense mutations, which are a severe type of base substitution, result in a stop codon in a position where there was not one before, which causes the Docket No.: 021025 / WO premature termination of protein synthesis and can result in a complete loss of function in the finished protein.

[0291] Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by lie, Leu by He, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

[0292] “Highly stringent hybridization conditions” are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e. , 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: Tm = 81.5 °C + 16.6(log [Na+]) + 0.41 (fraction G / C content) - 0.63(% formamide) - (600 / I). Furthermore, the Tm of a DNA: DNA hybrid is decreased by 1-1 ,5°C for every 1 % decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

[0293] Host cells can be transformed using a variety of standard techniques known to the art Docket No.: 021025 / WO

[0294] (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988.

[0295] Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile- mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transformed cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.

[0296] Conservative Substitutions Docket No.: 021025 / WO

[0297] Positively Charged

[0298] (Basic): K R H

[0299] Negatively Charged

[0300] (Acidic): D E

[0301] Exemplary nucleic acids that may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate Docket No.: 021025 / WO with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0302] Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0303] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807- 15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401 -423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several Docket No.: 021025 / WO siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.

[0304] GENOME EDITING

[0305] As described herein, gene and / or protein expression signals can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing.

[0306] As described herein, activity, signals, expression, or function can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing (e.g., upregulate, downregulate, overexpress, underexpress, express (e.g., transgenic expression), knock in, knock out, knockdown).

[0307] Processes for genome editing are well known; see e.g., Aldi 2018 Nature Communications 9(1911 ). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0308] For example, genome editing can comprise CRISPR / Cas9, CRISPR-Cpf1 , TALEN, or ZNFs. Adequate blockage of gene / protein expression / signaling by genome editing can result in protection from autoimmune or inflammatory diseases.

[0309] As an example, clustered regularly interspaced short palindromic repeats (CRISPR)ZCRISPR-associated (Cas) systems are a new class of genome-editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR / Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9 (thus, a (N)2oNGG target DNA sequence). This results in a double-strand break three nucleotides upstream of the NGG motif. The double strand break instigates either non-homologous end-joining, which is error-prone and conducive to frameshift mutations that knock out gene alleles, or homology-directed repair, which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. Thus, genomic editing, for example, using CRISPR / Cas systems could be useful tools for therapeutic applications to Docket No.: 021025 / WO target cells by the removal or addition of signals (e.g., activate (e.g., CRISPRa), upregulate, overexpress, downregulate).

[0310] For example, the methods described herein can comprise a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein.

[0311] GENE THERAPY AND GENOME EDITING

[0312] Gene therapies can include inserting a functional gene with a viral vector. Gene therapies are rapidly advancing.

[0313] There has recently been an improved landscape for gene therapies. For example, in the first quarter of 2019, there were 372 ongoing gene therapy clinical trials (Alliance for Regenerative Medicine, 5 / 9 / 19).

[0314] Any vector known in the art can be used. For example, the vector can be a viral vector selected from retrovirus, lentivirus, herpes, adenovirus, adeno-associated virus (AAV), rabies, Ebola, lentivirus, or hybrids thereof. Gene therapy strategies. Docket No.: 021025 / WO

[0315] Gene therapy can allow for the constant delivery of the enzyme directly to target organs and eliminates the need for weekly infusions. Also, correction of a few cells could lead to the enzyme being secreted into the circulation and taken up by their neighboring cells (cross-correction), resulting in widespread correction of the biochemical defects. As such, the number of cells that must be modified with a gene transfer vector is relatively low.

[0316] Genetic modification can be performed either ex vivo or in vivo. The ex vivo strategy is based on the modification of cells in culture and transplantation of the modified cell into a patient. Cells that are most commonly considered therapeutic targets for monogenic diseases are stem cells. Advances in the collection and isolation of these cells from a variety of sources have promoted autologous gene therapy as a viable option.

[0317] The use of endonucleases for targeted genome editing can solve the limitations presented by the usual gene therapy protocols. These enzymes are custom molecular scissors, allowing cutting DNA into well-defined, perfectly specified pieces, in virtually all cell types. Moreover, they can be delivered to the cells by plasmids that transiently express the nucleases, or by transcribed RNA, avoiding the use of viruses.

[0318] FORMULATION

[0319] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0320] The term "formulation" refers to preparing a drug in a form suitable for administration Docket No.: 021025 / WO to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0321] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc., may also be used.

[0322] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0323] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0324] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or Docket No.: 021025 / WO attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0325] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently, affect the occurrence of side effects. Controlled- release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0326] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

[0327] THERAPEUTIC METHODS

[0328] Also provided is a process of treating, preventing, or reversing at least one of inflammation, inflammation-induced tissue injury, and / or ischemic stroke in a subject in need thereof via administration of a therapeutically effective amount of anti-inflammatory peptide(s).

[0329] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing at least one of inflammation, inflammation-induced tissue injury, and / or ischemic stroke. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human Docket No.: 021025 / WO subject.

[0330] Generally, a safe and effective amount of anti-inflammatory peptide(s) is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of anti-inflammatory peptide(s) described herein can substantially inhibit, slow the progress of, or limit the development of at least one of inflammation, inflammation-induced tissue injury, and / or ischemic stroke.

[0331] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0332] When used in the treatments described herein, a therapeutically effective amount of anti-inflammatory peptide(s) can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to reduce inflammation, reduce or prevent inflammation-induced tissue injury, and / or prevent or treat ischemic stroke.

[0333] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0334] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0335] The specific therapeutically effective dose level for any particular subject will depend Docket No.: 021025 / WO upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw- Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0336] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes reversing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or a physician.

[0337] Administration of anti-inflammatory peptide(s) can occur as a single event or over a time course of treatment. For example, anti-inflammatory peptide(s) can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment Docket No.: 021025 / WO from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0338] Treatment in accord with the methods described herein can be performed prior to or before, concurrent with, or after conventional treatment modalities for at least one of inflammation, inflammation-induced tissue injury, and / or ischemic stroke.

[0339] An anti-inflammatory peptide (or peptides)can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, an anti-inflammatory peptide (or peptidesjcan be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of an anti-inflammatory peptide, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of anti-inflammatory peptide(s), an antibiotic, an anti-inflammatory, or another agent. An anti-inflammatory peptide (or peptidesjcan be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, an anti-inflammatory peptide (or peptidesjcan be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent.

[0340] Active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition in a patient. For example, the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in treating the disease in a human or another animal, such as the model systems shown in the examples and drawings.

[0341] An effective dose range of a therapeutic can be extrapolated from effective doses determined in animal studies for a variety of different animals. In general, a human equivalent dose (HED) in mg / kg can be calculated in accordance with the following formula (see e.g., Reagan-Shaw et al., FASEB J., 22(3):659-661 , 2008, which is incorporated herein by reference):

[0342] HED (mg / kg) = Animal dose (mg / kg) x (Animal Km / Hurnan Km)

[0343] Use of the Km factors in conversion results in more accurate HED values, which are based on body surface area (BSA) rather than only on body mass. Km values for humans and various animals are well known. For example, the Km for an average 60 kg human (with a Docket No.: 021025 / WO

[0344] BSA of 1 .6 m2) is 37, whereas a 20 kg child (BSA 0.8 m2) would have a Km of 25. Km for some relevant animal models are also well known, including: mice Km of 3 (given a weight of 0.02 kg and BSA of 0.007); hamster Km of 5 (given a weight of 0.08 kg and BSA of 0.02); rat Km of 6 (given a weight of 0.15 kg and BSA of 0.025) and monkey Km of 12 (given a weight of 3 kg and BSA of 0.24).

[0345] Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are peculiar to each individual. Nonetheless, a calculated HED dose provides a general guide. Other factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment, and the potency, stability, and toxicity of the particular therapeutic formulation.

[0346] The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a subject may be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the subject and on the route of administration. These factors may be determined by a skilled artisan. The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. The dosage may be adjusted by the individual physician in the event of any complication.

[0347] In some embodiments, the anti-inflammatory peptide(s) may be administered in an amount from about 1 mg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg, or about 1 mg / kg to about 25 mg / kg, or about 1 mg / kg to about 15 mg / kg, or about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg, or about 3 mg / kg. In some embodiments, anti-inflammatory peptide(s) such as those described herein may be administered in a range of about 1 mg / kg to about 200 mg / kg, or about 50 mg / kg to about 200 mg / kg, or about 50 mg / kg to about 100 mg / kg, or about 75 mg / kg to about 100 mg / kg, or about 100 mg / kg.

[0348] The effective amount may be less than 1 mg / kg / day, less than 500 mg / kg / day, less than 250 mg / kg / day, less than 100 mg / kg / day, less than 50 mg / kg / day, less than 25 mg / kg / day or less than 10 mg / kg / day. It may alternatively be in the range of 1 mg / kg / day to 200 mg / kg / day.

[0349] In other non-limiting examples, a dose may also comprise from about 1 micro- gram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body Docket No.: 021025 / WO weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above.

[0350] CELL THERAPY

[0351] Cells generated according to the methods described herein can be used in cell therapy. Cell therapy (also called cellular therapy, cell transplantation, or cytotherapy) can be a therapy in which viable cells are injected, grafted, or implanted into a patient in order to effectuate a medicinal effect or therapeutic benefit. For example, transplanting T-cells capable of fighting cancer cells via cell-mediated immunity can be used in the course of immunotherapy, grafting stem cells can be used to regenerate diseased tissues, or transplanting beta cells can be used to treat diabetes.

[0352] Stem cell and cell transplantation has gained significant interest by researchers as a potential new therapeutic strategy for a wide range of diseases, in particular for degenerative and immunogenic pathologies.

[0353] Allogeneic cell therapy or allogenic transplantation uses donor cells from a different subject than the recipient of the cells. A benefit of an allogeneic strategy is that unmatched allogenic cell therapies can form the basis of "off the shelf" products.

[0354] Autologous cell therapy or autologous transplantation uses cells that are derived from the subject’s own tissues. It could also involve the isolation of matured cells from diseased tissues, to be later re-implanted at the same or neighboring tissues. A benefit of an autologous strategy is that there is limited concern for immunogenic responses or transplant rejection.

[0355] Xenogeneic cell therapies or xenotransplantation uses cells from another species. For example, pig derived cells can be transplanted into humans. Xenogeneic cell therapies can Docket No.: 021025 / WO involve human cell transplantation into experimental animal models for assessment of efficacy and safety or enable xenogeneic strategies to humans as well.

[0356] ADMINISTRATION

[0357] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0358] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0359] Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0360] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus Docket No.: 021025 / WO requiring only a fraction of a systemic dosage.

[0361] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve taste of the product; or improve shelf life of the product.

[0362] SCREENING

[0363] Also provided are screening methods.

[0364] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 MW, or less than about 1000 MW, or less than about 800 MW) organic molecules or inorganic molecules including but not limited to salts or metals.

[0365] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0366] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for Docket No.: 021025 / WO commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example, ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).

[0367] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0368] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0369] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict the bioavailability of a compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0370] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less Docket No.: 021025 / WO than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and 0 atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

[0371] KITS

[0372] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to the anti-inflammatory peptides of the present disclosure or precursors thereof. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0373] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral nonreacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0374] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and / or may be supplied as an electronic- readable medium or video. Detailed instructions may not be physically associated with the Docket No.: 021025 / WO kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0375] A control sample or a reference sample as described herein can be a sample from a healthy subject or sample, a wild-type subject or sample, or from populations thereof. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects or a wild-type subject or sample. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0376] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sam brook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0377] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0378] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of Docket No.: 021025 / WO reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0379] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0380] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0381] 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 with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0382] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and Docket No.: 021025 / WO claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0383] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0384] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0385] EXAMPLES

[0386] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0387] EXAMPLE 1 - NEUTROPHIL CRACR2A PROMOTES NEUTROPHIL RECRUITMENT IN STERILE INFLAMMATION AND ISCHEMIC STROKE.

[0388] In this example, studies were performed to demonstrate that neutrophil CRACR2A promotes neutrophil recruitment to sites of sterile inflammation, such as ischemic stroke. Blocking the STIM1-CRACR2A interaction can mitigate inflammation and consequent tissue injury.

[0389] INTRODUCTION Docket No.: 021025 / WO

[0390] While neutrophils are important for innate immune responses, their excessive recruitment to sites of inflammation causes tissue damage. Neutrophil recruitment on activated endothelial cells (ECs) is a sequential process, including rolling, arrest, crawling, and transmigration, mediated by the interactions between neutrophil and EC adhesion molecules. After neutrophil rolling, mediated by the interaction between selectins and their ligands, neutrophils adhere and crawl on ECs, which are mainly controlled by aL[32 and aM[32 integrin, respectively. The final step of neutrophil transmigration requires various molecules, including [32 integrin, CD31 , and junctional adhesion molecules. Although neutrophil surface receptors required for neutrophil recruitment have been well identified, how their functions are regulated remains poorly understood.

[0391] As a large family of small GTPases, Rab GTPases are involved in intracellular vesicle trafficking in numerous cell types and contribute to the pathology of cancer, neurodegenerative diseases, diabetes, and immune diseases. Among them, Ca2+release- activated Ca2+channel regulator 2A (CRACR2A, also known as Rab46) exists in both long (CRACR2A-L, around 90-kDa) and short (CRACR2A-S, around 45-kDa) forms. CRACR2A- L contains two EF-hands, a coiled-coil domain, a Pro-rich domain, a Rab GTPase domain, and a C-terminal prenylation site, whereas CRACR2A-S only contains two EF-hands and a coiled-coil domain. In T cells, both CRACR2A-L and CRACR2A-S promote store-operated Ca2+entry (SOCE) and activate the Ca2+-nuclear factor of activated T-cells (NFAT) signaling pathway. Importantly, a recent study identified a patient with biallelic mutations in the CRACR2A gene, who exhibited reduced cytokine production and SOCE in T cells with the development of an immunodeficiency disorder. However, the role of neutrophil CRACR2A in inflammation is unknown.

[0392] Using intravital microscopy (IVM) of myeloid-specific cracr2a conditional knockout (CKO) mice, it was demonstrated that neutrophil cracr2a is crucial for neutrophil recruitment on peripheral microvessels in TNF-a-induced systemic and local inflammation. CRACR2A-deficient neutrophils or differentiated human neutrophil-like (dHL-60) cells exhibit defects in Ca2+mobilization and the ligand-binding function of [32 integrin after agonist stimulation. Overexpression of CRACR2A rescues all defects in CRACR2A KO dHL-60 cells. Treatment of neutrophils with a palmitoylated 20-mer derived from the stromal interaction molecule 1 (STIMI )-binding region in CRACR2A recapitulates the defects found in cracr2a- deficient neutrophils. Four-dimensional IVM of cerebral microvessels of mice following focal Docket No.: 021025 / WO brain ischemia reveals that neutrophil cracr2a increases the emergence of a unique subpopulation of circulating neutrophils with upregulated aM|32 integrin and enhanced migratory activity, promoting neutrophil infiltration into brain tissue and contributing to brain damage. These results demonstrate that neutrophil CRACR2A is a crucial Ca2+regulator required for neutrophil adhesive and migratory functions in sterile inflammation, such as vasculitis and ischemic stroke. Blocking the CRACR2A-STIM1 interaction may be a therapeutic strategy to mitigate excessive neutrophil recruitment in inflammatory conditions.

[0393] RESULTS

[0394] Genome-wide association studies (GWAS) identify the association between various single-nucleotide polymorphisms (SNPs) of the CRACR2A gene and cardiovascular or inflammatory diseases

[0395] A recent study reported a patient with biallelic mutations in the CRACR2A gene who has both missense variants (A>G in 12:3788175 and G>T in 12:3765501 ) and a stop-gained variant (G>T in 12:3763526). The patient has an immunodeficiency disorder characterized by T cell dysfunction resulting from impaired SOCE and JNK activation. To further examine whether CRACR2A is involved in human diseases, databases were searched, including the National Health Genome Research Institute - European Bioinformatics Institute (NHGRI-EBI) catalog of genome-wide association studies (GWAS), Common Metabolic Diseases Knowledge Portal, IEU OpenGWAS project, and UKBiobank PheWeb for SNPs in the CRACR2A gene. In addition to an immunodeficiency disorder observed in a patient, various CRACR2A SNPs were associated with cardiovascular diseases, such as cerebral infarction and heart failure, or inflammatory conditions, such as dermatitis herpetiformis and periodontitis (Table 3). This finding suggests possible links between CRACR2A and cardiovascular or inflammatory diseases.

[0396] Table 3: SNPs in genes encoding CRACR2A protein are associated with Docket No.: 021025 / WO

[0397] Neutrophil cracr2a promotes neutrophil recruitment on ear and cremaster venules in TNF-a-induced sterile inflammation

[0398] Preclinical and clinical studies suggest that CRACR2A is involved in T cell signaling and related immune responses. To investigate whether neutrophil CRACR2A regulates neutrophil recruitment in vascular inflammation, wild-type littermate control (WT) (cracr2afl / fl) and myeloid-specific cracr2a CKO mice (cracr2afl / fl;Lyz'cre+ / _) were generated (FIG. 1A). Immunoblotting showed that mouse and human neutrophils expressed both CRACR2A-L and CRACR2A-S, with a different pattern (FIG. 1 B). The expression of cracr2a was markedly reduced but not completely deleted in neutrophils from the CKO mice (Figure 1 C). Partial knockdown of cracr2a was also observed in monocytes from the CKO mice, while cracr2a expression was not altered in platelets from the CKO mice. The targeted gene was correctly deleted in CKO neutrophils as confirmed by rt-PCR (FIG. 7A, FIG. 7B). Compared to WT mice, cracr2a CKO mice had similar blood counts (Table 4), and cracr2a deficiency did not affect the expression of STIM1 , aM and (32 integrin subunits, or talinl in neutrophils (FIG. Docket No.: 021025 / WO

[0399] 1 C). To determine the physiological role of neutrophil cracr2a in acute inflammation, IVM was performed with WT and cracr2a CKO mice subjected to an intraperitoneal injection of TNF- a (FIG. 1 D). Compared to WT mice, the CKO mice exhibited increased neutrophil rolling but decreased adhesion, crawling, and transmigration on inflamed ear venules (FIG. 1 E, FIG. 1 F, FIG. 1G, FIG. 1 H, FIG. 11). When the mice were treated with an intrascrotal injection of TNF-a, cracr2a deficiency did not affect neutrophil rolling but significantly reduced neutrophil adhesion, crawling, and transmigration on cremaster venules (FIG. 1 J, FIG. 1 K, FIG. 1 L, FIG. 1 M, FIG. 1 N, FIG. 10). Further analyzed of the slow rolling speed of neutrophils showed that cracr2a deficiency increased the speed on the ear but not cremaster venules (FIG. 8A, FIG. 8B). The discrepant result in neutrophil rolling on two different venules may be due to surgery- induced occlusion or bleeding of some branches in the cremaster muscle, which may alter microcirculation. Since multiple venules were analyzed in one mouse, the correlation between measurements from the same mouse was examined by a linear mixed model, and the results were consistent with those shown in FIG. 1 H (FIG. 9A).

[0400] Table 4: CBCs in WT littermate control and cracr2a CKO mice.

[0401] A recent study has shown that platelet glycoprotein VI is crucial for plateletneutrophil aggregation and neutrophil recruitment in lipopolysaccharide-induced acute lung injury. In contrast, it was previously reported that depletion of platelets did not affect neutrophil recruitment in postcapillary venules in TNF-a-induced vascular inflammation. It was found that platelets adhered to adherent neutrophils in ear and cremaster venules of TNF-a-challenged WT mice, and the number and percentage of platelet-bound neutrophils were significantly reduced in CKO mice (FIG. 10B, FIG. 10C). To further investigate whether platelets contribute to neutrophil recruitment, WT and CKO mice were treated with control IgG or anti-GPIba antibodies that rapidly clear circulating platelets (FIG. 11 A, FIG. 11 B). One hour later, the mice were treated with TNF-a, followed by IVM. The absence of platelets did not affect neutrophil rolling, adhesion, crawling, and transmigration on ear (FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E) or cremaster venules (FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E) in Docket No.: 021025 / WO

[0402] WT and CKO mice. Consistent with previous reports, these results suggest that neutrophil recruitment to postcapillary venules in TNF-a-induced sterile inflammation results from neutrophil-EC but not neutrophil-platelet interactions. Overall, these results indicate that neutrophil cracr2a promotes neutrophil recruitment in TNF-a-induced systemic and local inflammation.

[0403] Neutrophil cracr2a enhances the ligand-binding function of |32 integrin by promoting Ca2+mobilization

[0404] To study how neutrophil cracr2a promotes neutrophil recruitment in acute inflammation, it was investigated whether cracr2a regulates the expression and function of neutrophil adhesion receptors. Deficiency of neutrophil cracr2a reduced the amount of cellsurface aMf32 integrin in the presence of 1 mM CaCI? after stimulation with N-formyl- methionyl-leucyl-phenylalanine (fMLP) or A23187 (FIG. 2A) but did not affect the expression of L-selectin, aL[32 integrin, junctional adhesion molecule A, CD31 (platelet EC adhesion molecule-1 ), or P-selectin glycoprotein ligand-1 (PSGL-1 ) (FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E). In addition, the binding of soluble fibrinogen (FG), a ligand of aM[32 integrin, to stimulated neutrophils was significantly decreased by cracr2a deficiency (FIG. 2B). Since neutrophil aM[32 integrin is stored in secretory granules and translocates to the plasma membrane during cell activation, these results suggest that neutrophil cracr2a promotes degranulation of aM[32 integrin and its ligand-binding function during cell activation. It was further confirmed that deficiency of neutrophil cracr2a reduced the activity of gelatinase (a marker for specific and gelatinase granules) in the releasate after agonist stimulation (FIG. 15). To determine whether decreased FG binding is a direct consequence of decreased surface levels of aM[32 integrin, FG binding (FIG. 2B) was normalized to the surface level of aMp2 integrin (FIG. 2A). Deficiency of neutrophil cracr2a still reduced FG binding (FIG. 2C), implying that the cracr2a-promoted ligand-binding function of aM|32 integrin is likely due to its enhanced activation.

[0405] The final step in [32 integrin activation is the binding of talin 1 to the cytoplasmic tail of the [32 integrin subunit. Using immunoprecipitation, it was found that talinl -[32 binding was enhanced in fMLP-activated neutrophils, which was reduced by cracr2a deficiency (FIG. 2D). This result suggests that neutrophil cracr2a promotes [32 integrin activation, and the regulatory function of neutrophil cracr2a is unlikely to be limited to aM[32 integrin. Docket No.: 021025 / WO

[0406] Cytosolic Ca2+facilitates the ligand binding function of (32 integrin. CRACR2A has been shown to promote SOCE by interacting with both STIM1 and Orail during T cell activation. It was observed that neutrophil cracr2a (cracr2a-l) rapidly interacted with STIM1 after fMLP stimulation (FIG. 2E). Deficiency of neutrophil cracr2a significantly reduced Ca2+mobilization after stimulation with fMLP or A23187 as measured by a Fura-2-based Ca2+assay (FIG. 2F, FIG. 2G, FIG. 2I). A similar result was obtained with thapsigargin (TG) treatment, which increases the cytosolic Ca2+level by blocking sarco / endoplasmic reticulum Ca2+ATPase (FIG. 2H, FIG. 2I). Also performed were Ca2+assays using another Ca2+dye (FLIPR) with rapid action and a higher sensitivity than Fura-2. Deficiency of neutrophil cracr2a impaired both Ca2+release and influx after stimulation with fMLP or A23187 and diminished spontaneous Ca2+accumulation and influx induced by TG (FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E). The baseline of peak Ca2+levels in cracr2a-deficient neutrophils was slightly but constantly lower than that of WT neutrophils (FIG. 16F). The peak levels of Ca2+release and influx after agonist stimulation were reduced by cracr2a deficiency (FIG. 16F, FIG. 16G). The decrease in agonist-induced Ca2+mobilization in cracr2a-deficient neutrophils did not result from the alteration in dye-loading or quenching since WT and cracr2a-deficient neutrophils were labeled with calcein-AM equally (FIG. 17).

[0407] Since deficiency of neutrophil cracr2a reduces neutrophil crawling and transmigration on the inflamed endothelium and impairs [32 integrin function, neutrophil spreading and polarization was measured under shear and static conditions. When fMLP-stimulated WT and cracr2a-deficient neutrophils were perfused over the surfaces coated with a mixture of ICAM-1 and E-selectin under 1 dyne / cm2, it was observed that cracr2a deficiency significantly reduced the number of adherent neutrophils and their spreading area (FIG. 2 J, FIG. 2K, FIG. 2L). It is reported that the newly formed F-actin localizes at the leading edge, while phosphorylated myosin light chain (p-MLC) is at the uropod. To examine whether cracr2a regulates neutrophil polarization, WT and cracr2a-deficient neutrophils were plated on FG-coated surfaces in the presence of fMLP. It was found that cracr2a deficiency reduced the spreading area of adherent neutrophils and impaired the separation of F-actin and p-MLC localization as determined by the Pearson coefficient (FIG. 2M, FIG. 2N, FIG. 20). Overall, these results suggest that neutrophil cracr2a regulates Ca2+content in the endoplasmic reticulum and promotes both Ca2+release and STIM1 -Orail -induced SOCE, facilitating (32 integrin-mediated neutrophil spreading and polarization. Docket No.: 021025 / WO

[0408] Studies have shown that STIM1 deletion markedly reduces SOCE required for the production of reactive oxygen species (ROS) in adherent neutrophils. However, deficiency of neutrophil cracr2a did not affect ROS production after stimulation with fMLP or phorbol 12- myristate 13-acetate (PMA) (FIG. 18C), suggesting that given the moderate defect in Ca2+signaling in cracr2a-deficient neutrophils, neutrophil cracr2a does not fully control STIM1- Orail -induced SOCE.

[0409] CRACR2A in differentiated HL-60 cells is critical for cytosolic Ca2+mobilization and 02 integrin activation

[0410] It is known that dHL-60 cells are similar to human neutrophils in their morphology and function. Thus, CRACR2A KO HL-60 cells were generated by editing 16 nucleotides (-16 indel) in the exon 6 of the CRACR2A gene using CRISPR / Cas9 (FIG. 3A). After differentiation, CRACR2A deletion did not affect the viability of dHL-60 cells and the expression of other proteins, such as STIM1 and aM02 integrin (FIG. 3B, FIG. 3C). CRACR2A-S was deleted in the KO dHL-60 cells, although a band was detected at a molecular weight (86-kDa) lower than CRACR2A-L (FIG. 3C). Next-generation sequencing was further performed for two WT and CRACR2A KO HL-60 cell lines and confirmed CRISPR / Cas9-mediated deletion of the target site in the KO HL60 cells (F08) (FIG. 19A, FIG. 19B, FIG. 19C). Since CRACR2A exon 6 targeted by the single-guide RNA contains 96 base pairs (amino acids 255-286), this result suggests that CRISPR / Cas9-mediated frameshift indels cause in-frame exon skipping, as reported previously.

[0411] Flow cytometry using WT (E04) and CRACR2A KO dHL-60 cells (F08) and conformation-specific antibodies revealed that CRACR2A deletion reduced the binding of antibodies against total aM02 integrin (ICRF44), but not antibodies against activated aM02 (CBRM1 / 5) or activated 02 (mAb24), to unstimulated dHL-60 cells (FIG. 3D, FIG. 3E, FIG. 3F), implying that the surface level of aM02 integrin in cultured dHL-60 cells is spontaneously increased by degranulation, but the integrin is not fully activated. CRACR2A deletion did not affect the surface amount of PSGL-1 on unstimulated or stimulated dHL-60 cells (FIG. 20). It was found that deletion of CRACR2A significantly diminished the binding of all antibodies to dHL-60 cells after stimulation with fMLP or A23187. Further, CRACR2A deletion impaired Ca2+mobilization in dHL-60 cells after treatment with fMLP, A23187, or TG (FIG. 3G, FIG. Docket No.: 021025 / WO

[0412] 3H, FIG. 3I, FIG. 3J). These results suggest that CRACR2A deletion in dHL-60 cells recapitulates the defects observed in cracr2a-deficient neutrophils.

[0413] Next, a vector expressing N-terminal mCherry or m Cherry-tagged CRACR2A-L or CRACR2A-S was transfected into the KO dHL-60 cells to investigate whether the aforementioned impairments could be reversed (FIG. 3K). Compared to the control, overexpression of either CRACR2A-L or CRACR2A-S rescued the defects in Ca2+mobilization and [32 integrin activation in the KO cells (FIG. 3L, FIG. 3M, FIG. 3N, FIG. 30, FIG. 3P, FIG. 3Q). These results suggest that like mouse neutrophil cracr2a, human CRACR2A has a similar function promoting Ca2+mobilization and [32 integrin activation.

[0414] Palmitoylated 20-mer derived from the STIM1 binding region in CRACR2A inhibits Ca2+mobilization and 2 integrin activation in mouse and human neutrophils

[0415] Although neutrophil cracr2a interacted with STIM1 during cell activation and increased Ca2+mobilization and 02 integrin function, the lack of pharmacological inhibitors of CRACR2A hindered us from exploring its therapeutic potential. Since a study has shown that the coiled-coil region in CRACR2A binds to STIM1 , an eight N-terminal palmitoylated 20- mers (P1 to P8) and one 16-mer (P9) were synthesized, covering the STIM1 binding region in mouse and human CRACR2A (FIG. 21 A, FIG. 21 B, FIG. 21 C, Table 1 , Table 2). When mouse neutrophils were treated with each peptide (10 pM), peptides 1 , 4, 5, 7, and 8 affected cell viability as measured by propidium iodide staining (FIG. 4A). Peptide 2 significantly reduced Ca2+mobilization in mouse neutrophils after stimulation with fMLP, A23187, or TG (FIG. 4B, FIG. 4C, FIG. 4D), and peptide 6 impaired Ca2+mobilization in neutrophils stimulated with fMLP, but not other agonists. Peptides 1 , 2, 3, 5, 6, and 7 reduced the surface level of aM[32 integrin in fMLP-stimulated neutrophils (FIG. 4E), whereas only peptide 2 significantly inhibited soluble FG binding (FIG. 4F). It was observed that pretreatment with peptide 2 but not 3 significantly blocked cracr2a-STIM1 interaction in fMLP-stimulated neutrophils (FIG .4G). Based on these results, peptides 2 and 3 were further tested in a flow chamber assay in which peptide-treated mouse neutrophils were stimulated with fMLP and perfused over a monolayer of TNF-a-stimulated mouse pulmonary vein ECs under a venous shear condition (2 dyne / cm2). Peptide 2 significantly increased the number of rolling neutrophils and reduced the number of neutrophil crawling and transmigration on the EC monolayer, while peptide 3 had no effect (FIG. 4H, FIG. FIG. 4I, FIG. 4J, FIG. 4K). These Docket No.: 021025 / WO results suggest that peptide 2 derived from the amino acids 221-240 in the coiled-coil region of mouse cracr2a blocks cracr2a-STIM1 binding, Ca2+mobilization, and neutrophil adhesion and migration on activated ECs. See Table 1 and Table 2.

[0416] To test the effect of peptide 2 in vivo, first an adoptive transfer experiment was performed. C57BL / 6 mouse neutrophils with 85% purity were labeled with calcein-AM or CellTracker Deep Red dye and then incubated with vehicle (green) or 10 pM peptide 2 (red) (FIG. 4L, FIG. 4M). Then, an equal number of vehicle- or peptide-treated neutrophils were mixed and injected into recipient C57BL / 6 mice 2 hours after an intraperitoneal injection of TNF-a. When IVM was performed 1 hour later, it was found that compared to the vehicle control, pretreatment with peptide 2 significantly reduced neutrophil adhesion and crawling, but not rolling, on ear venules in TNF-a-challenged mice (FIG. 4N, FIG. 40, FIG. 4P, FIG. 4Q). The discrepancy between cracr2a CKO mice and the peptide 2 effect on neutrophil rolling may result from the partial inhibition of neutrophil cracr2a by P2. Next, the in vivo effect of peptide 2 was tested on neutrophil recruitment. The peptide was intravenously injected into C57BL / 6 mice right before or 2.5 hours after an intraperitoneal injection of TNF-a. When IVM was conducted 3 hours after TNF-a injection, either treatment did not show any inhibitory effects on neutrophil recruitment on ear venules (FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D), implying that peptide 2 is likely rapidly degraded in circulation.

[0417] To test peptides in human neutrophils, peripheral neutrophils were isolated and confirmed 90-92% purity (FIG. 23A, FIG. 23B). When human neutrophils were pretreated with peptides (10 pM) derived from the coiled-coil region in human CRACR2A, peptides 1 , 4, 5, and 7 impaired cell viability (FIG. 4R). Peptides 1 and 2 significantly decreased Ca2+mobilization in human neutrophils after stimulation with fMLP, A23187, or TG (FIG. 4S, FIG. 4T, FIG. 4U). In contrast, peptides 3, 7, and 9 impaired Ca2+mobilization in response to only a specific agonist. Pretreatment with peptide 2 inhibited the surface level, but not activation, of aM|32 integrin in unstimulated human neutrophils (FIG. 4V, FIG. 4W). Only peptide 2 significantly reduced the surface level and activation of aM[32 integrin and inhibited (32 integrin activation in fMLP-stimulated human neutrophils, whereas peptide 3 and 7 showed the inhibitory effect on the surface level or activation of aM|32 integrin but not [32 integrin activation (FIG. 4V, FIG. 4W, FIG. 4X). The inhibitory effect of peptide 2 on the surface level and activation of aM[32 integrin was significantly greater upon agonist stimulation (>85% reduction) compared to the unstimulated condition (28-36% reduction). Since the sequence Docket No.: 021025 / WO of peptide 2 in mouse and human CRACR2A is identical, these results suggest that palmitoylated peptide 2 inhibits [32 integrin activation and may have therapeutic potential for reducing neutrophil adhesive function.

[0418] Neutrophil cracr2a promotes neutrophil migratory function and contributes to brain damage following focal brain ischemia in mice

[0419] Studies have shown that deletion of hematopoietic cell STIM1 or Orail or loss of aM[32 or aL[32 integrin protects against cerebral ischemia in mice. Since neutrophil cracr2a enhances Ca2+mobilization, [32 integrin function, and neutrophil recruitment in sterile inflammation (FIG. 1 C, FIG. 2I, FIG. 2L), it was investigated whether neutrophil cracr2a contributes to brain damage in a mouse model of ischemic stroke induced by transient middle cerebral artery occlusion (tMCAO). Compared to WT mice, cracr2a CKO mice exhibited improved neurological deficits as assessed by the Bederson score and grip strength test and reduced infarct volume 24 hours after tMCAO (FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D).

[0420] To further study whether neutrophil cracr2a regulates neutrophil recruitment in ischemic stroke, 4D confocal IVM was performed using a cranial window in WT and cracr2a CKO mice. After a 4-week recovery from the cranial window surgery, mice were challenged with tMCAO and conducted IVM every 6 hours for 24 hours (FIG. 5E). Rolling and crawling neutrophils were visualized by anti-Ly-6G and anti-aM(32 antibodies on pial and cortical microvessels as shown in the maximal intensity projection (30 frames for 1 minute) of realtime imaging 24 hours after tMCAO (FIG. 5F). Compared to WT mice, CKO mice exhibited a significant reduction in the surface level of oM(32 integrin in intravascular Ly-6G+neutrophils. The minimal numbers of rolling and crawling neutrophils were detected until 12 hours after tMCAO (FIG. 5G, FIG. 5H). While the number of rolling neutrophils was similar between WT and CKO mice, the number of crawling neutrophils was significantly less in CKO mice 24 hours after tMCAO. Single neutrophil behaviors were further analyzed by unsupervised clustering and uniform manifold approximation and projection (UMAP) of 6 categories, including track length, average speed, aM[32 intensity, displacement length, straightness, and duration, in WT and cracr2a CKO mice 24 hours after tMCAO (FIG. 5I, FIG. 5J, FIG. 24A, FIG. 24B, FIG. 24C). Three distinct subpopulations were identified in 434 intravascular neutrophils. Clusters 1 and 3 represented crawling neutrophils with a distinct expression of aM|32 integrin (aM(32highin cluster 1 and aM(32l0Win cluster 3), and cluster 2 represented Docket No.: 021025 / WO rolling neutrophils with a broad expression of aM[32 integrin. When neutrophil behaviors were dissected between WT and cracr2a CKO mice, clusters 1 and 2 were reduced, but cluster 3 was increased in CKO mice (FIG. 5I). Compared to WT mice, CKO mice exhibited a significant reduction in the surface level of aM[32 integrin in all subsets of neutrophils (FIG. 5K), which is consistent with reduced crawling (FIG. 5H). It was also investigated whether intravascular aM[32highneutrophils interact with platelets by injecting anti-CD42c antibodies. No platelets stably attached to adherent aM[32highneutrophils on pial and cortical microvessels 18 and 24 hours after tMCAO (FIG. 25A, FIG. 25B), likely due to the faster blood flow compared to that in ear and cremaster venules.

[0421] When transmigrated neutrophils were analyzed 24 hours after tMCAO, the number was significantly less in cracr2a CKO mice compared to WT mice (FIG. 5L, FIG. 5M). Also conducted were single-cell behavioral analysis of 313 transmigrated neutrophils and identified 2 distinct subsets (FIG. 5N, FIG. 50). Compared to cluster 2, cluster 1 had more migratory aM[32highneutrophils (FIG. 50). Compared to WT mice, transmigrated neutrophils in CKO mice had significantly lower levels of aM|32 integrin in only cluster 1 (FIG. 5P). The average speed of transmigrated neutrophils in clusters 1 -2 was slower in the CKO mice compared to WT mice (FIG. 5Q). These results suggest that neutrophil cracr2a promotes the migratory function of intravascular and transmigrated neutrophils by upregulating the surface level of aM 2 integrin, contributing to brain damage in ischemic stroke.

[0422] Neutrophil cracr2a promotes the emergence of a unique subpopulation of circulating neutrophils with enhanced migratory activity in ischemic stroke

[0423] Since aM[32 integrin has been used as a marker for circulating proinflammatory neutrophils under inflammatory conditions, such as sickle cell disease, proposed here is that neutrophil cracr2a enhances the emergence of oM[32highmigratory neutrophils in the circulation in ischemic stroke, thereby regulating neutrophil heterogeneity. To study this, blood was collected from WT and cracr2a CKO mice 24 hours after tMCAO and labeled neutrophils with Ly-6G, aM[32, L-selectin, and FG. Compared to WT mice, cracr2a CKO mice exhibited increased levels of L-selectin and decreased levels of aM[32 integrin on the surface of peripheral neutrophils (FIG. 6A, FIG. 6B, FIG. 6C). In addition, the CKO mice exhibited a significantly lower percentage of aMp2high / L-selectinl0Wneutrophils but a higher percentage of aM[32l0W / L-selectinhighneutrophils compared to WT mice (FIG. 6D, FIG. 6E). To further Docket No.: 021025 / WO investigate whether aM[32 integrin is activated in circulating neutrophils after the tMCAO challenge, the result were analyzed with aM|32 integrin and FG (FIG. 6F). FG binding to circulating neutrophils and the percentage of aM[32high / FGhighneutrophils were significantly reduced in the CKO mice (FIG. 6G, FIG. 6H). These results suggest that cracr2a promotes the surface level and activation of aM|32 integrin in circulating neutrophils and enhances neutrophil migratory activity in ischemic stroke.

[0424] In a steady state, neutrophils are present in a young or aged state. Aged neutrophils are known to upregulate aMp2 integrin and CXCR4 and downregulate L-selectin on the surface. To determine whether neutrophil cracr2a contributes to heterogeneity in a steady state, blood was collected from WT and cracr2a CKO mice every 4 hours. The percentage of young (Ly-6G+ / aM[32l0W / L-selectinhigh) and aged (Ly-6G+ / aM[32high / L-selectinl0W) neutrophils was not different between WT and CKO mice (FIG. 6I, FIG. 6J), suggesting that neutrophil cracr2a does not influence neutrophil aging in a steady state.

[0425] DISCUSSION

[0426] A recent study identifies one immunodeficient patient with biallelic mutations in CRACR2A. In the present study, it was found that various SNPs in the CRACR2A gene are linked to cardiovascular or inflammatory diseases. The IVM using myeloid-specific cracr2a CKO mice demonstrates that neutrophil cracr2a promotes neutrophil recruitment to ear and cremaster venules in acute sterile inflammation. In addition, 4D cerebral IVM in a mouse model of ischemic stroke and single neutrophil behavioral analysis suggests that neutrophil cracr2a increases the number of a unique subpopulation of circulating neutrophils with enhanced migratory activity and facilitates neutrophil infiltration into the brain tissue, contributing to brain damage. Using neutrophils and dHL-60 cells deficient in CRACR2A, it was found that neutrophil CRACR2A positively regulates Ca2+mobilization and enhances the ligand-binding function of [32 integrin in inflammatory conditions. Importantly, the study using a palmitoylated 20-mer provides evidence that blocking the CRACR2A-STIM1 interaction may be a therapeutic strategy for attenuating neutrophil adhesive and migratory functions in inflammatory diseases.

[0427] Rab GTPases have approximately 60 family members in humans and regulate intracellular vesicle formation, transport, fission, and fusion. CRACR2A (Rab46) has long and short forms with different expression patterns in various cell types. In addition to two EF- Docket No.: 021025 / WO hands and a coiled-coil domain in CRACR2A-S, CRACR2A-L has a Pro-rich domain interacting with Vav1 , which is crucial for the accumulation of CRACR2A-containing vesicles at the immunological synapse, and also contains the C-terminal prenylated GTPase domain that regulates its degradation. It is reported that T cells express both long and short forms. Srikanth et al. reported that siRNA-mediated knockdown of CRACR2A-S in Jurkat cells reduces STIM1 / Orai1 -mediated SOCE and that CRACR2A-L is localized in subsynaptic vesicles that translocate to the immunological synapse after ligation of T cell receptors, activating downstream signaling. In ECs, only CRACR2A-L is expressed and promotes tube formation without affecting Ca2+mobilization. Subsequent studies have shown that CRACR2A-L localizes to P-selectin-negative Weibel-Palade bodies (WPB) and interacts with dynein, resulting in the trafficking of a subpopulation of WPBs to the microtubule-organizing center in histamine-stimulated ECs. Using polyclonal anti-CRACR2A antibodies that recognize amino acids 1 -200 of CRACR2A, it was found that both long and short forms of CRACR2A are expressed in human and mouse neutrophils, with a different pattern. Despite the incomplete deletion of neutrophil cracr2a-l and cracr2a-s, the CKO mice exhibit significant defects in neutrophil recruitment in TNF-a-induced acute inflammation. In CRACR2A KO dHL-60 cells, the CRISPR / Cas9 approach causes in-frame exon skipping (amino acids 255- 286 in the coiled-coil region) in the CRACR2A gene and produces a 86-kDa protein (FIG. 3C, FIG. 18C). Nevertheless, the finding that the KO dHL-60 cells exhibit the same defects observed in cracr2a-deficient neutrophils and those defects are rescued by overexpression of either CRACR2A-S or CRACR2A-L suggests the 86-kDa protein is likely non-functional in promoting Ca2+mobilization.

[0428] Previous studies using STIM1 or STIM2 KO or CKO mice have shown that deletion of STIM1 abrogates SOCE and impairs ROS production during neutrophil activation and that deletion of neutrophil STIM2 affects SOCE after stimulation with low concentrations of agonists and reduces production of cytokines but not ROS, degranulation, or phagocytosis. It is reported that both Orail and Orai2 are critical for SOCE, degranulation, ROS production, and phagocytosis. In HeLa cells overexpressing CRACR2A, STIM1 , and Orai isoforms, CRACR2A-S interacts with STIM1 and all three Orai isoforms. It was found that neutrophil cracr2a interacts with STIM1 and that deficiency of neutrophil cracr2a causes a significant but moderate reduction in Ca2+mobilization and (32 integrin function without affecting ROS production after agonist stimulation. These results suggest that neutrophil cracr2a does not Docket No.: 021025 / WO fully control STIM1 / Oral-induced SOCE and that neutrophil cracr2a-regulated cytosolic Ca2+levels may specifically contribute to neutrophil adhesive function. In line with this speculation, the IVM indicates that neutrophil cracr2a promotes neutrophil recruitment to the inflamed ear and cremaster venules (FIG. 1 E, FIG. 1 K).

[0429] Because of the critical role of STIM1 / 0rai1 -induced SOCE in human pathophysiology, CRAC channel inhibitors are being developed to treat autoimmune or inflammatory conditions. However, the fact that various loss-of-function mutations in STIM1 or Orail are directly associated with infections, autoimmunity, muscular hypotonia, and ectodermal dysplasia suggests that STIM1 or Orail inhibitors may have adverse effects, such as impaired immune responses. A previous study has implied that the coiled-coil region in CRACR2A interacts with the coiled-coil and PEST domains in STIM1. Identify herein was a palmitoylated 20-mer (amino acids 221 -240 in mouse cracr2a and 227-246 in the human counterpart) that inhibits cracr2a-STIM1 binding, Ca2+mobilization, [32 integrin activation, and neutrophil crawling and transmigration on the inflamed EC monolayer under a venous shear condition. The results in the adoptive transfer experiment (FIG. 4L, FIG. 4M, FIG. 4N, FIG. 40, FIG. 4P, FIG. 4Q) demonstrate that blocking the CRACR2A-STIM1 interaction is a therapeutic strategy to reduce excessive neutrophil recruitment to sites of inflammation. Additional studies will investigate the core sequence of peptide 2 that binds to STIM1 and develop small-molecule inhibitors targeting the CRACR2A-STIM1 interface.

[0430] Neutrophils are heterogeneous and have both beneficial and detrimental roles in inflammation. It is reported that pro-inflammatory neutrophils have an increased level of aM[32 integrin in inflammatory conditions, such as sickle cell disease. Although targeting tissue-damaging, proinflam matory neutrophils identified by specific markers may be effective for attenuating inflammatory conditions without impairing immune responses, this concept has not been studied well because there are challenges in identifying neutrophil subpopulations responsible for tissue damage, and the molecular mechanism regulating spatiotemporal neutrophil heterogeneity remains unclear. A recent study using the individual leukocyte behavioral analysis of 4D IVM provides insight into persistent cell identities and their immune profiles in inflammation. The single neutrophil behavioral analysis of 4D IVM demonstrates that neutrophils are present in distinct subsets inside and outside the cerebral microvessels in ischemic stroke. However, there is a limitation to the study. Since live imaging of the infarct core is not feasible, the IVM was conducted in the pial and cortical microvessels Docket No.: 021025 / WO within the penumbra region. Thus, neutrophil behaviors may differ between two regions due to differences in blood flow rates, structures and sizes of blood vessels, and the time courses of neutrophil infiltration. Nevertheless, the results clearly show that neutrophil cracr2a enhances the emergence of highly migratory neutrophils by increasing the surface level and activation of aM[32 integrin, thereby promoting neutrophil infiltration into brain tissue and exacerbating brain injury.

[0431] Overall, the studies using cracr2a CKO mice, CRACR2A KO dHL-60 cells, and an inhibitory peptide provide strong evidence that neutrophil CRACR2A is crucial for neutrophil adhesive and migratory functions in sterile inflammation and ischemic stroke.

[0432] METHODS

[0433] Reagents

[0434] Rabbit control IgG, rat IgG, fMLP, A23187, human FG, horseradish peroxidase, luminol, and PMA were purchased from Sigma-Aldrich (St. Louis, MO). TG was from Cayman Chemical (Ann Arbor, Ml). Recombinant mouse TNF-a, propidium iodide solution, isotype control IgGs, rat lgG2a, BV421 -, Alexa Fluor 488-, PE-, or Alexa Fluor 647-conjugated antimouse Ly-6G (1A8), Alexa Fluor 488-, PE-, or Alexa Fluor 647-conjugated anti-mouse CD31 (MEC13.3), PE- or Alexa Fluor 700-conjugated anti-mouse aM (M1 / 70), PE-conjugated antihuman aM[32 (ICRF44), PE-conjugated antibodies against activated human aM[32 (CBRM1 / 5), FITC-conjugated anti-human (32 (mAb24), FITC-conjugated anti-mouse aL (M17 / 4), PE-conjugated anti-mouse Jam-A (27-9), Alexa Fluor 700-conjugated anti-CD66b (G10F5), PE-conjugated anti-human CD14 (HCD14), FITC-conjugated anti-human CD15 (MMA), APC-conjugated anti-human PSGL-1 (KPL-1 ), and FITC- or BV421 -conjugated antimouse L-selectin (MEL-14) antibodies were purchased from BioLegend (San Diego, CA). A PE-conjugated anti-mouse CD162 (2PH1 ) antibody was from BD Biosciences (San Jose, CA). DyLight 649-conjugated anti-mouse CD42c and anti-mouse GPIba (R300) antibodies were purchased from emfret Analytics (Wurzburg, Germany). Protein A / G agarose beads and 4% paraformaldehyde solution were obtained from Santa Cruz Biotechnology (Dallas, Texas). A rabbit monoclonal anti-STIM1 and rabbit polyclonal anti-p-MLC antibodies were from Cell Signaling Technology (Danvers, MA). Rabbit polyclonal antibodies against both mouse and human CRACR2A, (32, or talinl were purchased from Proteintech (Rosemont, IL). Also, rabbit polyclonal anti-CRACR2A antibodies were obtained from Abclonal (Woburn, Docket No.: 021025 / WO

[0435] MA). HRP-monoclonal mouse anti-rabbit IgG was from Jackson ImmunoResearch (West Grove, PA). Rabbit monoclonal anti-[3-actin antibodies were purchased from LI-COR (Lincoln, NE) and Novus Biological (Centennial, CO). Rabbit polyclonal anti-aM and anti- mCherry antibodies, Halt™ protease inhibitor cocktail, Neon™ transfection system kits, Alexa Fluor 488-conjugated FG, SuperScript III Reverse Transcriptase, SuperFi II 2x Platinum Green master mix, EnzChek™ Gelatinase / Collagenase Assay kit, calcein AM, CellTracker Deep Red dye, Alexa Fluor 488-conjugated phalloidin, and Alexa Fluor 647- conjugated goat anti-rabbit IgG antibodies were obtained from Thermo Fisher Scientific (Waltham, MA). FLIPR Ca2+5 assay kit and Fura-2 QBT Ca2+kit were from Molecular Devices (San Jose, CA). The N-terminal palmitoylated peptides (eight 20-mers and one 16- mer) derived from the coiled-coil region of mouse and human CRACR2A were synthesized by Peptide 2.0 (Chantilly, VA).

[0436] Animals

[0437] WT (C57BL / 6J) and Lyz-cre mice (6-8 weeks old) were obtained from The Jackson Laboratory. Cracr2aflox / floxmice with a background of C57BL / 6. WT littermate control (cracr2aflox / flox:Lyz‘cre- / ") and myeloid-specific cracr2a CKO mice (cracr2aflox / flox;Lyz’cre+ / ") were generated by crossing Lyz-Cre mice (B6.129P2-Lyz2 / J, JAX: 004781 ) with cracr2aflox / floxmice. All mice were housed in a 12-hour light / dark cycle in a temperature and humidity- controlled room, with ad libitum access to standard chow and water. Both the research team and the veterinary staff monitored animals daily. Animal health was monitored by weight (weekly), food and water intake, and general assessment of animal activity (daily). Power analysis and prior experience were used to guide the number of animals in each group to achieve statistical significance based on the anticipated effects of the treatments and genetic manipulations.

[0438] Experimental groups and randomization

[0439] Age-matched (8-14 weeks old) male and female WT and Cracr2a CKO mice were used in all studies except IVM of cremaster venules, in which age-matched male mice were used. Animals were randomly assigned to control and experimental groups in all experiments. When measuring various parameters or performing assays, the order in which samples were processed or analyzed was randomized. Animal studies reported in this paper comply with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). Docket No.: 021025 / WO

[0440] Inclusion and exclusion criteria

[0441] For stroke and cerebral IVM, mice were carefully monitored after surgery by researchers to recognize any signs of decreased activity, ruffled coat, hunched posture, labored breathing, unsteady gait, hind limb paralysis, and head tilt / spinning. If any of the above signs were noticed during monitoring and unable to be alleviated by a veterinarian, mice were humanely euthanized using CO2 asphyxiation. Of the 43 mice used for these studies, 4 mice died shortly after or did not recover from tMCAO. In addition, 11 mice were excluded from the study because they had inflammation on the cranial window or did not show vessel occlusion or reperfusion as assessed by blood flow rates as described in the method of tMCAO.

[0442] Search for CRACR2A SNPs associated with cardiovascular or inflammatory diseases

[0443] The National Health Genome Research Institute - European Bioinformatics Institute (NHGRI-EBI) catalog of GWAS and UKBiobank PheWeb were used to examine the association of CRACR2A (Gene ID: 84766) SNPs with cardiovascular or inflammatory disease conditions using a P-value of less than 10'7. The identified SNPs were further confirmed by their chromosome location, variation type, and allele variation in NCBL

[0444] Isolation of neutrophils and monocytes

[0445] Mouse and human neutrophils were isolated. Bone marrow cells were obtained from the femur and tibia of WT and cracr2a CKO mice by flushing with Hank's Balanced Salt Solution (HBSS) containing 20 mM Hepes, pH 7.4, and 0.1 % BSA. The cell suspension was filtered through 40 pm nylon meshes and centrifuged at 300 g at 4°C for 10 minutes. After resuspension with HBSS, cells were laid on the top of 59% Percoll solution and centrifuged at 1 ,200 g at 4°C for 30 minutes. The upper cell layer was collected as monocytes. The cell pellet was resuspended with HBSS, and red blood cells were lysed using a lysis buffer (Sigma-Aldrich). Neutrophils were centrifuged at 700 g at 4°C for 5 minutes and resuspended with HBSS (1 x 107cells / ml). Human neutrophils were isolated using a Percoll density gradient. Sodium citrate-treated human blood was mixed with an equal volume of 3% dextran and sedimented for 15 minutes. The supernatant (leukocytes-containing layer) was laid on Docket No.: 021025 / WO the top of a discontinuous Percoll gradient (55% and 74%) and centrifuged at 430 g at 12°C for 1 hour. The polymorphonuclear cells were collected at the interface of the two Percoll layers, resuspended with HBSS buffer, and then centrifuged at 300 g at 4°C for 10 minutes. After RBC lysis, neutrophils were resuspended as described above. As determined by flow cytometry, the purity of mouse and human neutrophils was 80-85% (FIG. 4M) and 90-92% (FIG. 19C), respectively. All healthy donors provided informed consent. The collection and use of blood samples for laboratory analysis were approved by the Institutional Review Board of Washington University School of Medicine.

[0446] IVM

[0447] WT and cracr2a CKO mice were injected intravenously with Alexa Fluor 488- conjugated anti-Ly-6G antibodies (0.2 pg / g mouse) and Alexa Fluor 647-conjugated anti- CD31 antibodies (0.8 pg / g mouse) and then intraperitoneally with murine TNF-a (40 ng / g mouse). Three hours later, the mice were anesthetized with ketamine (125 pg / g mouse) and xylazine (12.5 pg / g mouse). The left ear was immediately mounted on a customized heating glass for the initiation of imaging. Mice were placed on a warming pad to maintain body temperature at 37 °C during intravital imaging. Images were captured in the inflamed cremaster and ear venules with a diameter of 25-40 pm. The numbers of rolling, adherent, crawling, and transmigrated / transmigrating neutrophils were determined in an area of 0.32 mm2for 10 minutes. The numbers of rolling, adherent, and crawling neutrophils intral um inal ly were normalized to the inside area of blood vessels. Adherent neutrophils were defined as those that were stationary for >30 seconds, whereas crawling neutrophils were defined as those that were displaced by 10 pm during 1 minute. Due to the vessel heterogeneity, three to five different venules were monitored in one mouse.

[0448] In some experiments, neutrophils from C57BL / 6 mice were labeled with either 500 nM calcein AM or CellT racker Deep Red dye. Calcein AM- or Deep Red-labeled neutrophils were incubated with 0.1 % DMSO or 10 pM of an inhibitory peptide, respectively, for 30 minutes at 37 °C. Recipient C57BL / 6 mice (male 8-10 weeks old) were treated with an intraperitoneal injection of TNF-a (40 ng / g mouse). Two hours later, vehicle or peptide-treated neutrophils were mixed with an equal number of 3 x 106cells in 100 pl and intravenously injected with 100 pl along with PE-conjugated anti-CD31 antibodies (0.8 pg / g mouse) through a tail vein Docket No.: 021025 / WO into the recipient mice. One hour later, neutrophils were visualized on venules on the left ear and analyzed as described above.

[0449] To test the effect of an inhibitory peptide in vivo, C57BL / 6 mice (male 8-10 weeks old) were injected with anti-Ly-6G and anti-CD31 antibodies as described above. Then, vehicle (1.3% DMSO in saline) or the peptide (13.3 pg / g mouse) in 100 pl saline was injected via a tail vein into the mice right before or 2.5 hours after an intraperitoneal injection of TNF-a (40 ng / g mouse). IVM was performed 3 hours after TNF-a injection to visualize neutrophil recruitment on ear venules.

[0450] To induce local inflammation in cremaster venules, WT and cracr2a CKO mice were injected intravenously with Alexa Fluor 488-conjugated anti-Ly-6G antibodies (0.2 pg / g mouse) into mice and then intrascrotally with murine TNF-a (20 ng / g body weight (BW)). Two and a half hours after TNF-a injection, mice were anesthetized as described above, and the cremaster tissue was mounted on the imaging plate. Fifteen minutes after mounting, the cremaster tissue was imaged for 30-60 minutes. Since some cremaster venules had several branches, those with non-occluded branches were analyzed. Three to four different venules were monitored in each mouse.

[0451] To visualize neutrophils on cremaster and ear venules, fluorescence and bright-field images were recorded under a 20x objective lens (W Plan-Apochromat 20x, NA 1 .0, waterimmersion, Zeiss, Pleasanton, CA) using a Zeiss Axio examiner Z1 microscope system with a Yokogawa confocal spinning disk (CSU-W1 ) equipped with a four-stack laser system (405 nm, 488 nm, 561 nm, and 637 nm wavelengths) or a high-power LED light source (X-Cite XLED1 6-Channel Light Source (390-415, 450-495, 505-545, 540-600, 615-655, and 735- 780 nm), Excelitas Technologies, Waltham, MA). Images were collected with a high-speed, high-resolution camera (2304 x 2304 pixel format, ORCA-Fusion BT sCMOS, Hamamatsu, Shizuoka, Japan) and analyzed using SlideBook, version 6.0 (Intelligent Imaging Innovations, Denver, CO).

[0452] To visualize platelet-bound neutrophils on ear venules in TNF-a-induced systemic inflammation, WT and cracr2a CKO mice were injected intravenously with anti-Ly-6G and anti-CD31 antibodies as described above and DyLight 649-conjugated anti-CD42c antibodies (0.2 pg / g mouse) for platelet labeling. To observe platelet-bound neutrophils on cremaster venules in TNF-a-induced local inflammation, WT and cracr2a CKO mice were Docket No.: 021025 / WO injected intravenously with Alexa Fluor 488-conjugated anti-Ly-6G antibodies and with DyLight 649-conjugated anti-CD42c antibodies (0.2 pg / g mouse).

[0453] To examine the effect of platelet depletions on ear and cremaster venules, WT and cracr2a CKO mice were intravenously injected with non-immune rat IgG or anti-GPIba antibodies (R300, 0.5 pg / g mouse). One hour later, a complete blood count (CBC) was measured. Neutrophils, platelets, and endothelial cells were labeled as described above. IVM was performed 3 hours after intraperitoneal TNF-a injection to visualize neutrophils and platelets.

[0454] For cerebral intravital imaging, mice bearing a cranial imaging window (see cranial imaging window surgery below) were mounted on a custom-built stereotaxic plate after anesthetizing with 1 .2-1 .5% isoflurane. Neutrophils on cerebral microvessels were visualized and monitored for 30-60 minutes. Mice were on a warming pad to maintain the body temperature at 37°C during imaging. Neutrophils, aM[32, and ECs were labeled with Alexa Fluor 647-conjugated anti-Ly-6G (0.1 pg / g mouse), PE-conjugated anti-aMp2 (0.02 pg / g mouse), and Alexa Fluor 488-conjugated anti-CD31 antibodies (0.4 pg / g mouse), respectively. To detect platelet-bound neutrophils on cerebral microvessels of mice subjected to tMCAO, WT and cracr2a CKO mice were injected intravenously with anti-aM[32 and anti- CD31 antibodies as described above, and with BV421 -conjugated anti-Ly-6G (0.1 pg / g mouse) and DyLight 649-conjugated anti-CD42c antibodies (0.2 pg / g mouse). The numbers of intraluminal platelet-bound neutrophils were normalized to the inside area of blood vessels. Time-lapse imaging of neutrophils was performed for 10 minutes at 6, 12, 18, and 24 hours after tMCAO surgery. The numbers of rolling, crawling, and transmigrated / transmigrating neutrophils were determined in an area of 0.32 mm2for 10 minutes and normalized as described above.

[0455] RT-PCR

[0456] RNAs were isolated from mouse neutrophils with the RNeasy Plus mini kit (Qiagen) and used to generate cDNA with the Superscript III Reverse Transcriptase. RT-PCR was performed with OneTaq Quick-Load 2X Master Mix (New England Biolabs) and the following primers: mouse cracr2a exon3-4 forward: 5’-ACAGACTTGGAGCCCAGAAAG-3’ (SEQ ID NO: 1 ) and reverse: 5’- GAGGGCACATTCCAGTTCGT-3’ (SEQ ID NO: 2) Docket No.: 021025 / WO mouse cracr2a exon14-16 forward: 5’-CAAGCAGTCCTTCCTGTCGAT-3’ (SEQ ID NO: 3) and reverse: 5’-TCTCCTTGGCGAGTTGCTC-3’ (SEQ ID NO: 4) mouse gapdh forward: 5’-AGGTCGGTGTGAACGGATTTG-3’ (SEQ ID NO: 5) and reverse: 5 - TGTAGACCATGTAGTTGAGGTCA-3’ (SEQ ID NO: 6).

[0457] Flow cytometry

[0458] WT control and cracr2a-deficient neutrophils, human neutrophils, or dHL-60 cells (50 pl of 5 x 106cells / ml) were treated for 10 minutes with the following concentrations of fMLP or A23187: 5 pM fMLP or 0.5 pM A23187 for mouse neutrophils, 0.2 pM fMLP or 0.5 pM A23187 for human neutrophils, and 5 pM fMLP or 0.25 pM A23187 for dHL-60 cells in the presence of 1 mM CaCE and 1 mM MgCE. Cells were labeled using fluorescently labeled isotype control IgGs or antibodies against mouse L-selectin, mouse aM[32, mouse PSGL-1 , human PSGL-1 , activated human aM[32 (CBRM1 / 5), human aM[32 (ICRF44), or activated human P2 integrin (mAb24) for 20 minutes at room temperature. For FG binding assays, neutrophils were incubated with 30 pg / ml Alexa 488-labeled FG for 20 minutes at room temperature. To assess cell viability, mouse and human neutrophils were incubated with 0.1 mg / ml propidium iodide and FITC-conjugated anti-Ly6G for mouse neutrophils or Alexa Fluor 700-conjugated anti-CD66b, FITC-conjugated anti-CD15, and PE-conjugated anti-CD14 antibodies for human neutrophils for 15 minutes at room temperature. Flow cytometric analysis was performed using a CytoFlex flow cytometer (Beckman Coulter). Data were presented as the geometric mean fluorescence intensity (MFI) value.

[0459] To determine neutrophil heterogeneity in tMCAO-challenged mice, blood of WT and cracr2a CKO mice was collected at 23 hours after 1-hour tMCAO. After lysis of RBCs using a lysis buffer (Sigma-Aldrich), blood cells were centrifuged at 900 g at 4°C for 2 minutes and resuspended with HBSS (1 x 107cells / ml). Cells were labeled with fluorescently labeled control IgGs, antibodies against mouse L-selectin, aM[32, or Ly-6G, or Alexa Fluor 488- conjugated FG for 20 minutes at room temperature, followed by flow cytometry.

[0460] Gelatinase / collagenase activity assay

[0461] WT control and cracr2a-deficient neutrophils, 2 x 106cells / 100 pl, were stimulated for 10 minutes with 5 pM fMLP or 0.5 pM A23187. The releasates, 100 pl, were used to measure gelatinase activity using the EnzChek Gelatinase / Collagenase Assay Kit according to the Docket No.: 021025 / WO manufacturer’s instructions. The signal was read using a FlexStation3 microplate reader with an excitation wavelength of 495 nm and an emission wavelength of 515 nm. The activity was calculated using a standard curve of known concentrations of collagenase.

[0462] Immunoprecipitation

[0463] Neutrophil cracr2a or [32 was immunoprecipitated. Mouse neutrophils (500 pl of 1 x 107cells / ml) were treated with or without fMLP for 2, 5, or 10 minutes and then lysed with a lysis buffer (20 mM Hepes, pH 7.4, containing 1.5% NP-40, 50 mM ethylenediaminetetraacetic acid, 1.5% CHAPS, 0.5% sodium deoxycholate, 150 mM NaCI, proteinase inhibitor cocktail, 1 mM PMSF, 1 mM NaaVC and 1 mM NaF) on ice. Lysates were incubated with control IgG, anti-CRACR2A antibodies, or anti-|32 antibodies at 4°C, followed by incubation with protein A / G agarose beads for 1 hour at 4°C. Immunoblotting was performed using anti-STIM1 or anti-talin1 antibodies, and the band density was normalized to that of cracr2a or (32 , respectively.

[0464] Ca2+mobilization

[0465] WT and cracr2a-deficient neutrophils and dHL-60 cells (5 x 106cells / ml) were suspended in HBSS buffer. Cells were incubated with Fura-2 (Fura-2 QBT Calcium kit; Molecular Devices) for 30 minutes at 37 °C in the dark. Cytosolic Ca2+levels were measured after stimulation with 5 pM fMLP, 0.5 pM A23187, or 10 pM TG for mouse neutrophils, 0.2 pM fMLP, 0.5 pM A23187, or 10 pM TG for human neutrophils, or 5 pM fMLP, 0.25 pM A23187, or 5 pM TG for dHL-60 cells in the presence of 1 mM CaCh using a FlexStation3 microplate reader with an excitation wavelength of 340 and 380 nm and an emission wavelength of 510 nm. Ca2+mobilization was quantified by the area under the curve and expressed as a relative fluorescence unit. In some experiments, neutrophils were labeled with FLIPR (Molecular Devices) and then stimulated with 5 pM fMLP, 0.5 pM A23187, or 10 pM TG in the absence of Ca2+. After measuring Ca2+release, 1 mM CaCL was added to measure Ca2+influx. Cytosolic Ca2+levels were measured as described above (excitation: 485 nm and emission: 525 nm).

[0466] ROS production Docket No.: 021025 / WO

[0467] The wells of a 96-well plate were coated with 150 pg / ml human FG in 50 mM carbonate-bicarbonate buffer, pH 9.6, at 4°C overnight. WT and cracr2a-deficient neutrophils (4 x 106cells / 100 pl) in 20 mM Hepes, pH 7.4, containing 1 mM CaCl2 and 0.5 mM MgCl2) were mixed with 100 pM luminol and 8 ll / ml horseradish peroxidase. Then, cells were added to the wells and incubated at 37°C in a water bath for 10 minutes. After the addition of 20 pM fMLP or 50 nM PMA to the cells, light emission was recorded every 60 seconds using a FlexStation3 microplate reader at 37°C for 120 minutes.

[0468] Deletion or overexpression of CRACR2A in HL-60 cells

[0469] CRACR2A KO HL-60 cells were generated at the Genome Engineering & Stem Cell Center (GESC@MGI) at Washington University School of Medicine. HL-60 cells (American Type Culture Collection, Manassas, VA) were cultured in RPMI1640 media with a supplement of 10% fetal bovine serum and penicillin / streptomycin. CRACR2A was deleted in HL-60 cells by CRISPR / Cas9 using gRNA (5’-TCAAGCCCGCAGTCAAGAGCNGG-3’) (SEQ ID NO: 7). WT and CRACR2A KO HL-60 cells (2 x 105cells / ml) were differentiated by the addition of 1.5% DMSO to the media for 5-6 days before experiments. For the rescue experiment, CRACR2A plasmids (N-terminal flag-tagged CRACR2A-S (cat #79591 ) and N- terminal mCherry-tagged CRACR2A-L (cat #79593), Addgene, Watertown, MA) were purified by the QIAfilter plasmid maxi kit (Qiagen, Germantown, MD). An N-terminal mCherry- CRACR2A-S and N-terminal mCherry control plasmid were generated using DNA restriction enzymes (Xhol, TspMI, BspEI, and EcoRI, New England Biolabs) and Blunt / TA Ligase Master Mix (New England Biolabs) according to the manufacturer’s instructions. The control, CRACR2A-S, or CRACR2A-L vector was transfected into CRACR2A KO dHL-60 cells using a Neon transfection system according to the manufacturer’s instructions. Four hours after transfection, the overexpression was confirmed by immunoblotting and flow cytometry. After differentiation, cells were used for assays measuring Ca2+mobilization and integrin activation.

[0470] Next-generation sequencing

[0471] HL-60 cells were lysed in QuickExtract Solution from Lucigen (Madison, Wl), according to the manufacturer’s instructions. The target region is PCR amplified by tailed primers (gene-specific primer sequences below) appended with 5'- Docket No.: 021025 / WO

[0472] CACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 8) for forward and 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO: 9) for reverse to genomic-specific primer sequences (PCR1 ), which allows unique indexes and Illumina P5 / P7 adapter sequences to be added in a second round of PCR. PCR amplifications were performed with SuperFi II 2x Platinum Green master mix according to the manufacturer’s instructions. Indexing of the PCR1 product was performed by using 0.1 x volume from PCR1 with indexing primers (0.1 pM final concentration for each) and melting at 98 °C for 2 minutes, followed by five cycles of 98°C for 15 seconds, 60°C for 15 seconds, and 72 °C for 40 seconds. The lab generated 2 x 250 reads with the Illumina MiSeq platform at the Center for Genome Sciences and Systems Biology at Washington University in St. Louis. The extracted FASTQ files are analyzed using a Python-based alignment script.

[0473] Set 1 (832 bp) YM299.seq.F3: 5’-AGGAGACTCAGGAGCAAACG-3’ (SEQ ID NO: 10) YM299.seq.R3: 5’-GCCAGCTCCTGGTTAGTGAG-3’ (SEQ ID NO: 11 )

[0474] Set 2 (1 ,024 bp) YM299.seq.F3: 5 -AGGAGACTCAGGAGCAAACG-3’ (SEQ ID NO: 12)

[0475] YM299.seq.R2: 5’-CGAAGGTGCTTGTTCCTTTC-3’ (SEQ ID NO: 13)

[0476] Flow chamber assay

[0477] A monolayer of mouse pulmonary vein ECs (Cell Biologies, Chicago, IL) on gelatin- coated microfluidic chambers in a low shear plate (0-20 dyne / cm2, Cell Microsystems, Durham, NC) was stimulated with 10 ng / ml TNF-a for 3 hours. Neutrophils, 3 x 107cells / ml, were pretreated with vehicle (0.1 % DMSO) or 10 pM peptides for 20 minutes and then treated with 10 pM fMLP for 10 minutes at 37°C. After washing, 100 pl of neutrophil suspension was perfused over the EC monolayer for 5 minutes under 2 dyne / cm2using a BioFlux 1000z system (Fluxion Biosciences, Alameda, CA). There was a lag time of 5-10 seconds before recording to allow neutrophils to appear in the main chamber. Images were recorded under a I Ox / O.25 Ph1 objective lens (Zeiss, Pleasanton, CA) using an Axio Observer (Zeiss) with a high-power LED light source (4-Channels (385, 475, 555, and 630 nm), Colibri 5, Zeiss) and collected with a high-speed, high-resolution camera (2304 x 2304 pixel format, ORCA- Fusion BT sCMOS, Hamamatsu, Shizuoka, Japan). Rolling, crawling, and transmigrating / transmigrated neutrophils were counted in a field of 0.29 mm2and analyzed using BioFlux Montage Plus. Rolling neutrophils were identified as those that had a round Docket No.: 021025 / WO shape and moved on the EC monolayer, while crawling neutrophils were defined as those that polarized and slowly moved on the EC monolayer. Transmigrating neutrophils were identified as those that moved within the EC monolayer.

[0478] In some experiments, microfluidic chambers in a low shear plate were coated with 30 pg / ml of E-selectin and 15 pg / ml of ICAM-1 for 1 hour at 37°C. WT and cracr2a-deficient neutrophils, 2 x 1 o6cells / ml, were labeled with 500 nM calcein-AM and treated with or without 5 pM fMLP for 10 minutes at 37°C. After washing, a 200 pl neutrophil suspension was perfused for 10 minutes under 1 dyne / cm2using a BioFlux 1000z system. The area of adherent or spread neutrophils was measured using ImageJ 1 ,54f.

[0479] Confocal microscopy

[0480] To image polarized neutrophils, WT and cracr2a-deficient neutrophils, 5 x 105cells / ml, were plated on a Nunc™ Lab-Tek™ II Chamber Slide (Thermo Fisher Scientific) coated with 100 pg / ml of FG. After incubation for 10 minutes in the presence or absence of 5 pM fMLP at 37°C, adherent neutrophils were fixed with 2% paraformaldehyde. Then, cells were incubated with anti-p-MLC antibodies, and then Alexa Fluor 488-conjugated phalloidin for F- actin and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies for 1 hour. Neutrophils were imaged under a 63x objective lens (W Plan-Apochromat 63x, NA 1 .0, water-immersion) using a Zeiss Axio examiner Z1 microscope system with a Yokogawa confocal spinning disk (CSU-W1 ) equipped with a four-stack laser system and a high-speed, high-resolution camera as described in IVM. The Pearson coefficient of the location between F-actin and p-MLC was measured using Imaris software v9.02.

[0481] Effect of palmitoylated peptides

[0482] To test the inhibitory effect of palmitoylated peptides derived from the coiled-coil region of mouse and human CRACR2A, neutrophils were pretreated with vehicle (0.1% DMSO) or 10 pM of each peptide for 30 minutes at 37°C, followed by dye staining or agonist stimulation in all experiments.

[0483] TMCAO-induced ischemic stroke

[0484] TMCAO surgery was performed as described. Male mice (10-12 weeks old) were anesthetized with 1.2-1.5% isoflurane in an oxygen mixture and treated by an intraperitoneal Docket No.: 021025 / WO injection of buprenorphine-ER (0.1 pg / g mouse). The body temperature was maintained at 37°C throughout surgery using a warming pad. After exposure of the left common carotid artery, external carotid artery, and internal carotid artery, the MCA was occluded for 1 hour using silicon rubber-coated 6.0 nylon monofilament (Doccol Corporation, Sharon, MA). Cerebral blood flow was monitored using a laser Doppler perfusion system (PeriFlux System 5000, Perimed, Las Vegas, NV). A successful occlusion was indicated by a decrease in the regional cerebral blood flow to <10% of the baseline. Mice showing the blood flow to >10% of the baseline were excluded in this study. The filament was then removed, and blood flow was restored to the baseline. After 23 hours of reperfusion, the Bederson’s score and grip strength were measured. For the Bederson score, neurological deficits were scored according to the following system: 0, no observable deficit; 1 , forelimb flexion; 2, forelimb flexion and circling; 3, forelimb flexion, circling, and decreased resistance to lateral push; 4, death. For the grip strength test, tMCAO-challenged mice were placed on the iron grid connected to a strength meter (GT3, Bioseb, Pinellas Park, FL). Grip strength was assessed by recording maximal peak force (gram) as mice pulled out from the grid, serving as an indicator of neuromuscular function post-tMCAO. The test was performed 10 times with intervals of 3-5 minutes between each measurement, and each maximal peak force was normalized to BW. After assessing neurological behaviors, the brain was collected, and the 2 -mm brain slices were stained with 2% 2,3,5-triphenyltetrazolium chloride solution (Sigma- Aldrich) for 20 minutes, followed by fixation with 4% paraformaldehyde. Sections were scanned, and the infarct volumes were measured using Imaged 1 ,54f. This experiment was conducted blindly, with investigators uninformed about the identity of the mouse.

[0485] Cranial imaging window

[0486] A cranial imaging window was implanted for intravital imaging of the brain as described. To implant a cranial imaging window, mice were anesthetized with an intraperitoneal injection of ketamine and xylazine. The cranial bone was surgically exposed by skin incision, and the translucent connective tissue covering the cranial bone was removed. A circular-shaped hole (3 mm in diameter) in the cranial bone was drilled by the dental drill (Strong 207A, Saeshin, Irvine, CA) under a dissecting microscope. Using a fastcuring adhesive (Loctite 401. Henkel, Bridgewater, NJ), a 3 mm cover glass (Warner Instruments, Holliston, MA) was placed on the cranial bone to cover the exposed brain. Docket No.: 021025 / WO

[0487] Dental acrylic resin was used to cover the incision area around the cover glass. After a 4- week recovery period after implantation of an imaging window, mice with a clear view of the brain through the imaging window were used for intravital imaging.

[0488] Single-cell behavioral analysis

[0489] Single-cell behavioral analysis was performed as described. After cerebral intravital imaging after tMCAO, Ly-6G and aM|32-labeled neutrophil were analyzed behaviors by 6 categories, including track length, speed, aMp2 intensity, displacement length, duration, and straightness, for 10 minutes using Imaris software v9.02. A total of 434 intravascular neutrophils and 313 transmigrated neutrophils were uploaded to R Studio and used to generate UMAP plots. After clustering, density and doughnut plots were obtained by UMAP representation, and generated violin plots to compare the values of each cluster.

[0490] Statistics

[0491] Data was analyzed using GraphPad Prism (v10.2.2) software. When normality assumptions were confirmed, groups were compared using two-tailed Student’s f-test for two groups or one-way analysis of variance (ANOVA) and either Dunnett’s or Tukey’s test for three or more groups. When normality assumptions were rejected, non-parametric Mann- Whitney U test for two groups or ANOVA and Kruskal-Wallis tests were used. A P value less than 0.05 was considered significant.

Claims

Docket No.: 021025 / WOCLAIMSWhat is claimed is:1 . An anti-inflammatory composition comprising one or more peptides derived from a coiled-coil region of CRACR2A.

2. The anti-inflammatory composition of claim 1 , wherein at least one of the one or more peptides is palmitoylated.

3. The anti-inflammatory composition of claim 1 , wherein each of the one or more peptides is not less than a 10-mer peptide and is not more than a 50-mer peptide.

4. The anti-inflammatory composition of claim 1 , wherein at least one of the one or more peptides is selected from SEQ ID NO: 16-32.

5. The anti-inflammatory composition of claim 4, wherein at least one of the one or more peptides is SEQ ID NO: 17.

6. The anti-inflammatory composition of claim 1 , wherein at least one of the one or more peptides is selected from SEQ ID NO: 33-38.

7. A method of reducing inflammation in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of an anti-inflammatory composition comprising one or more peptides derived from a coiled-coil region of CRACR2A.

8. The method of claim 7, wherein the subject has sterile inflammation, sterile inflammation caused by ischemic stroke, sterile inflammation caused by vasculitis, or sterile inflammation caused by an autoimmune disease.

9. The method of claim 7, wherein at least one of the one or more peptides is palmitoylated.

10. The method of claim 7, wherein each of the one or more peptides is not less than a 10-mer peptide and is not more than a 50-mer peptide.Docket No.: 021025 / WQ11 . The method of claim 7, wherein at least one of the one or more peptides is selected from SEQ ID NO: 16-32.

12. The method of claim 7, wherein at least one of the one or more peptides is selected from SEQ ID NO: 33-38.

13. The method of claim 7, wherein the anti-inflammatory composition is administered at a concentration of at least 10 pM.

14. A method of blocking a STIM1 -CRACR2A interaction in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising one or more peptides derived from a coiled-coil region of CRACR2A.

15. The method of claim 14, wherein blocking the STIM1 -CRACR2A interaction in the subject reduces inflammation.

16. The method of claim 14, wherein blocking the STIM1-CRACR2A interaction in the subject reduces neutrophil migration to a site of inflammation, reduces neutrophil adhesion to endothelial cells (EC), inhibits aM|32 integrin activation, and blocks Ca2+ mobilization.

17. The method of claim 14, wherein at least one of the one or more peptides is selected from SEQ ID NO: 16-32.

18. The method of claim 14, wherein at least one of the one or more peptides is selected from SEQ ID NO: 33-38.

19. The method of claim 14, wherein the composition is administered at a concentration of at least 10 pM.

20. The method of claim 14, wherein the subject has sterile inflammation, sterile inflammation caused by ischemic stroke, sterile inflammation caused by vasculitis, or sterile inflammation caused by an autoimmune disease.

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