Compositions comprising nematode aminoacyl-TRNA synthetase and methods of using the same

Abstract

Disclosed are peptibodies, polynucleotides, cells comprising the polynucleotides, methods of using and making the peptibodies. The disclosed peptibodies comprise a nematode asparaginyl-tRNA synthetase, or a fragment thereof, linked to an antibody constant region. The nematode asparaginyl-tRNA synthetase may comprise Brugia malayi asparaginyl-tRNA synthetase (BmAsnRS), or a fragment thereof, e.g., the N-terminal fragment.

Description

Atty. Dkt. No. 650053.01247COMPOSITIONS COMPRISING NEMATODE AMINOACYL-TRNA SYNTHETASE AND METHODS OF USING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No.63 / 719,305 that was filed November 12, 2024, the entire contents of which are hereby incorporated by reference.SEQUENCE LISTING

[0002] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “650053_01247.xml” which is 5,187 bytes in size and was created on November 10, 2025. The sequence listing is electronically submitted via Patent Center and is incorporated by reference herein in its entirety.BACKGROUND

[0003] Chemokines and cancer biology are intertwined subjects. CXCL8 (interleukin-8, IL-8) is a proinflammatory chemokine that interacts with G protein coupled receptors (GPCRs), CXCR1 and CXCR2, on a wide variety of cells, and plays a key role in tumor biology, cancer pathogenesis, tumor angiogenesis and survival, and response to chemotherapy. CXCL8 has been proposed as a biomarker of tumor grow th, tyarrestins are multifunctional intracellular proteins with an ability to directly interact with GPCRs. P-arrestins contribute to multiple aspects of GPCR signaling, trafficking and downregulation, and thus like GPCRs, P-arrestins have emerged as important targets in new cancer therapies. Melanoma, lymphoma, prostate, lung, pancreatic, neural, head neck, breast and ovarian cancers all exhibit abnormal CXCL8 expression and therefore clinical trials have explored candidate CXCR1 / 2 antagonists as cancer treatments. Accordingly, novel therapies that target these pathways are needed in the art.SUMMARY

[0004] In an aspect of this disclosure, peptibodies are provided. In some embodiments, the peptibodies comprise a nematode asparaginyl-tRNA synthetase, or a fragment thereof, linked to an antibody constant region.

[0005] In an aspect of this disclosure, polynucleotides are provided. In some embodiments, the polynucleotides comprise a sequence encoding the disclosed peptibodies.

[0006] In an aspect of this disclosure, cells are provided. In some embodiments, the cells comprise the disclosed polynucleotides.Atty. Dkt. No. 650053.01247

[0007] In an aspect of this disclosure, methods are provided. In some embodiments, the methods comprise administering the disclosed peptibodies to a subject.

[0008] In an aspect of this disclosure, methods of treating a disease or disorder associated with CXCL8 expression in a subject in need thereof are provided. In some embodiments, the methods comprise administering the disclosed peptibodies to a subject in need thereof

[0009] In an aspect of this disclosure, methods of making a peptibody are provided. In some embodiments, the methods comprise cultivating the disclosed cells and enriching, isolating, or purifying the peptibody.

[0010] In an aspect of this disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise the disclosed peptibodies and a pharmaceutically acceptable excipient.BRIEF DESCRIPTION OF THE FIGURES

[0011] FIGs. 1A and IB show BRET bioluminescence (Y axis) is measured at different concentrations of BmAsnRS and CXCL8 using HEK cells expressing either CXCR1 (A) or CXCR2 (B). Data points represent means of quadruplicate replicates. Increasing concentrations of CXCL8 (black) decreases CXCR1 / 2 GPCR G protein subunit association as expected as the complex is activated, however BmAsnRS (blue) increases basal G protein subunit association, a feature of inverse agonism.

[0012] FIG. 2 shows BmAsnRS is a 548 amino acid (aa) homodimer with a classical 3 part modular structure typical of class II AARS: N terminus 88 (88aa), 33 aa unstructured linker connecting a 438 aa catalytic domain exhibiting anti parallel folds that bind ATP and is responsible for aminoacylation.

[0013] FIG. 3 shows a cartoon structure of BmAsnRS , indicating that the structured amino terminus binds to the receptor.

[0014] FIGs. 4A and 4B show Dali superimposed motifs of BmAsnRS (blue), CXCL8 (green) and SDF-1 (magenta). B shows the detailed correspondence between CXCL8 (green) and BmAsnRS (blue) structures (rotated by 90 degrees relative to panel A, focusing on contacts with the CXCR1 peptide (gray) bound in the NMR structure (PDB entry lilp;). Side chains are shown for all side chain contacts predicted between BmAsnRS and the CXCR1 peptide, based on the CXCL8 alignment. In the case of CXCL8, its N-loop is also involved in binding to CXCR1. A pattern was deduced from an alignment of 13 CXC chemokines including CXCL8 and CXCL12 (SDF-1, stromal derived factor 1 / melanoma stimulating factor).Atty. Dkt. No. 650053.01247

[0015] FIG. 5 shows (a) Modular design of TANGO constructs from Kroeze et al. “PRESTOTANGO: an open-source resource for interrogation of the druggable human GPCR-ome” Nat Struct Mol Biol. 2015 Apr 20;22(5):362-369. (top); blue arrowheads indicate Cla I sites, and green arrowheads indicate Age I sites. General scheme for the |3-arrestin (TANGO) recruitment assay (bottom). Upon activation of the GPCR by an agonist (1), 0-arrestin is recruited to the C-terminus of the receptor (2). This is followed by cleavage of the GPCR fusion protein at the TEV protease site (3). Cleavage results in the release of the tTA transcription factor (4), which, after transport to the nucleus, activates transcription of the luciferase reporter gene (5). (b) Surface expression of two selected TANGO constructs as shown by immunofluorescence using an anti-FLAG antibody. Concentration-response curves of a prototypical non-orphan GPCR, the neuromedin B receptor stimulated by neuromedin B (NMB) in the TANGO assay (c) and in a calcium-release assay (d); data are shown as the mean ± SEM of typical experiments done in quadruplicate. Curves were fitted using Graphpad Prism 5.0.

[0016] FIGs. 6A, 6B, and 6C show A) Plasmid 1 (Pl) encodes BmAsnRS N terminus (1-111). B) Plasmid P2 encodes 25kDa IgGFc. C) Plasmid P3 encodes a 38kDa protein representing IgGFc fused to BmAsnRS 1-111 (oval). A complete one-armed peptibody (63 kDa) combines proteins from plasmid 2 and plasmid 3. C) COOMASIE BLUE STAINED SDS PAGE GEL shows denatured BmAsnRS IgGFc peptibody (Lane 2) revealing the proteins from plasmids P2 (25 kDa) and P3 (38kDa). Lane 3 contains the complete non-denatured one-armed (one Fc fragment) 63 kDa peptibody. Lane 1 contains molecular weight markers in kDa.

[0017] FIG. 7 shows a comparison of BmAsnRS vs CXCL8 structure.

[0018] FIG. 8 shows a superimposition of the structures of BmAsnRS, CXCL8, and CXCL12.

[0019] FIG. 9 shows a schematic of GPCR structure and function and use of BRET assay to interrogate ligand / receptor interactions.

[0020] FIG. 10 shows observations related to BmsnRS versus CXCL8 activity and statements of how IL-8 and BmAsnRS elicit different responses at the same receptor

[0021] FIGs. 11 A and 11B show plasmid maps for the two components (chain 1, chain 2)of the synthetic peptibody. Co expression of these two proteins yields the BmAsnRS peptibody as shown in figure 6B. Chain 2 map (FIG. 11B) is the igGF Fc fragment without the parasite n terminus.Atty. Dkt. No. 650053.01247DETAILED DESCRIPTION

[0022] Disclosed herein are peptibodies, polynucleotides encoding the peptibodies, cells, methods of treating cancer in a subject in need thereof, methods of making a peptibody, and pharmaceutical compositions.Peptibodies

[0023] The inventors discovered that the asparaginyl-tRNA synthetase from Brugia malciyi acts as an inverse agonist of CXCR1 and CXCR2, which are the natural receptors for CXCL8. The inventors developed a peptibody comprising the asparaginyl-tRNA synthetase, or a fragment thereof, linked to an antibody constant region (FIGs. 6B and 6C).

[0024] Accordingly, in an aspect of this disclosure, peptibodies are provided. In some embodiments, the peptibodies comprise a nematode asparaginyl-tRNA synthetase, or a fragment thereof, linked to an antibody constant region.

[0025] A ‘'peptibody” as used herein, refers to a heterologous polypeptide linked to a portion of an antibody. Peptibodies may comprise a heterologous poly peptide linked to a constant region of the antibody. As used in the definition of “peptibody” above, “heterologous” refers to a polynucleotide or polypeptide that is not linked to an antibody, or a portion thereof in nature.

[0026] The peptibodies may be “one-armed” as show n in FIG. 6B, or “two-armed” comprising tw o nematode AsnRSs, e.g., BmAsnRS. Two-armed peptibodies comprise two binding moieties, e.g., the disclosed nematode AsnRSs, instead of a single binding moiety.

[0027] The nematode asparaginyl-tRNA synthetase may comprise or consist of SEQ ID NOs: 1, or a sequence with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%. at least about 98%, at least about 99%, or about 100% identity to SEQ ID NOs: 1.

[0028] The nematode asparaginyl-tRNA synthetase may comprise or consist of a portion of the tRNA synthetase, e.g., an N-terminal portion. As used herein, the “N-terminal portion” of a nematode asparaginyl-tRNA synthetase may refer to amino acids 1-79, 1-70, 1-60, 1-50, 1-40, 5-79, 10-79, 20-79, 30-79, 40-79, 50-79, or any subrange therein, relative to SEQ ID NO: 1, or a sequence with at least 80% identity to SEQ ID NO: 1. The N-terminal portion of the nematode asparaginyl-tRNA synthetase may comprise or consist of SEQ ID NO: 2 or a sequence with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%,Atty. Dkt. No. 650053.01247at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NO: 2.

[0029] The antibody constant region may comprise or consist of SEQ ID NO: 3 or a sequence with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NO: 3.

[0030] The peptibodies may comprise a linker between the nematode asparaginyl-tRNA synthetase and the antibody constant region, e.g., SEQ ID NO: 4, or a sequence with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. or about 100% identity to SEQ ID NO: 4.

[0031] Without being limited by any theory or mechanism, one hypothesis for how BmAsnRS acts as an inverse agonist is the large relative size of the protein compared to the native CXCL8 protein. Thus, the inventors developed a peptibody that comprises the N-terminus of BmAsnRS, which binds to CXCR1 or CXCR2 targets, and has a similar mass as the full length BmAsnRS protein. This may interrupt normal internalization of the ligand, i.e., CXCL8, and lead to the observed inverse agonism.Polynucleotides

[0032] In an aspect of this disclosure, polynucleotides are provided. In some embodiments, the polynucleotides comprise a sequence encoding a nematode asparaginyl-tRNA synthetase, or a fragment thereof, linked to an antibody constant region.

[0033] The polynucleotides may encode the disclosed peptibodies and further comprise a regulatory sequence operably linked to the sequence encoding the peptibodies. Exemplary regulatory sequences include, but are not limited to, promoters and enhancers.

[0034] As used herein, “operably linked” refers to a functional linkage between two or more sequences such that activity at or on one sequence affects activity at or on the other sequence(s). For example, an operable linkage between a polynucleotide of interest, e.g., a sequence encodingAtty. Dkt. No. 650053.01247a peptibody of the instant disclosure, and a regulatory' element (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest.Cells

[0035] The disclosed peptibodies may be expressed and produced in recombinant cells. Accordingly, in an aspect of this disclosure, cells are provided. In some embodiments, the cells comprise a polynucleotide comprising a sequence encoding a nematode asparaginyl-tRNA synthetase, or a fragment thereof, linked to an antibody constant region.

[0036] Suitable cells for the production of recombinant proteins are known in the art and include, but are not limited to, human embryonic kidney (HEK) 293 cells, Chinese hamster ovary (CHO) cells, orNIH323 cells.Methods

[0037] In an aspect of this disclosure, methods are provided. In some embodiments, the methods comprise administering the disclosed peptibodies to a subject. In other embodiments, the methods comprise contacting the disclosed peptibodies to a cell.

[0038] A “subject,” as used herein, may comprise a vertebrate, e.g., a mammal, e.g., a human, non-human primate, a companion animal, e.g., a dog, a cat, a working animal, e.g., a horse, a donkey, etc.

[0039] Administration may be performed by any suitable route, e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, intratumoral, intrathecal, etc., which may be determined by a physician. In an exemplary' embodiment, the peptibodies are administered intravenously or intratumorally.

[0040] The disclosed peptibodies may be administered in combination with at least one additional therapy, e.g., a chemotherapy, an immunotherapy, a surgery, a radiation therapy, or a hormone therapy. The immunotherapy may comprise an immune checkpoint inhibitor (ICI), several of which are known in the art including, but not limited to. an anti-PD-1 antibody, e.g., pembrolizumab, nivolumab, cemiplimab, or an anti-PD-Ll antibody, e.g., atezolizumab, avelumab, durvalumab, an anti-CTLA-4 antibody, e.g., ipilimumab and tremelimumab, an anti-LAG-3 antibody, e.g., relatlimab.Methods of treating diseases and disorders associated with CXCL8 expression

[0041] The disclosed peptibodies are inverse agonists of CXCR1 and CXCR2 (FIGs. 1A and IB), whose natural ligand is CXCL8. CXCL8 is dysregulated in certain diseases and disorders, e.g., cancer. Accordingly, methods of treating diseases and disorders associated with CXCL8 expression are provided. In some embodiments, the methods comprise administering theAtty. Dkt. No. 650053.01247disclosed peptibodies to a subject in need thereof. The disease or disorder associated with CXCL8 expression may include, but are not limited to, cancer, e.g., melanoma, lymphoma, prostate, lung, pancreatic, neural, head and neck, breast, or ovarian cancers.

[0042] The peptibodies may be administered to a '‘subject in need thereof’ which comprises a subject suffering from a disease or disorder associated with CXCL8 expression.

[0043] The disclosed peptibodies may be administered in a “therapeutically effective amount” which, as used herein, comprises an amount of the disclosed peptibodies that achieves at least one therapeutic goal, e.g., reduction in tumor size, reduction in overall tumor burden, reduction in tumor number, reduction in tumor or cancer grade, remission, or cure of the disease or disorder associated with CXCL8 expression.

[0044] The peptibodies may be administered in an amount from about 1.25 mg, 2.5 mg, 5 mg, 7.5 mg. 10 mg. 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, or more, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and / or maximal doses of the compounds may include doses falling within dose ranges having as endpoints any of these disclosed doses (e.g., 2.5 mg -200 mg).Methods of making a peptibody

[0045] In an aspect of this disclosure, methods of making the disclosed peptibodies are provided. In some embodiments, the methods comprise cultivating the disclosed cells comprising a polynucleotide comprising a sequence encoding a nematode asparaginyl-tRNA synthetase, or a fragment thereof, linked to an antibody constant region and enriching, isolating, or purifying the peptibody from the cells.

[0046] Methods of generating recombinant protein from genetically modified cells are known in the art. In certain embodiments, routine biochemical methods are used to enrich, isolate, or purify the peptibodies, e.g., high-performance liquid chromatography (HPLC), column chromatography, affinity chromatography, etc.

[0047] The disclosed peptibodies may comprise affinity ligands which permit affinity purification, e g., HIS tags, FLAG tags, etc., which are known and routine in the art.Atty. Dkt. No. 650053.01247Pharmaceutical compositions

[0048] Disclosed herein are pharmaceutical compositions comprising the disclosed peptibodies and at least one pharmaceutically acceptable excipient.

[0049] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized. Exemplar}7solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof.

[0050] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier mav be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0051] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and crosslinked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH 102. microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0052] Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PHI 01Atty. Dkt. No. 650053.01247and Avicel® PHI 02; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0053] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, com starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

[0054] Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0055] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route. For example, the pharmaceutical composition may be administered via oral, intravenous, intramuscular, subcutaneous, topical, and pulmonary route. Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders and granules. In some embodiments, the compounds are formulated as a composition for administration orally (e.g., in a solvent such as 5% DMSO in oil such as vegetable oil).

[0056] The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.

[0057] Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may’ be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0058] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueousAtty. Dkt. No. 650053.01247or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0059] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis.

[0060] Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

[0061] For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

[0062] Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (z.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

[0063] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.Atty. Dkt. No. 650053.01247

[0064] Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fdlers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0065] The present invention is described herein using several additional definitions, as set forth below and throughout the application.

[0066] Additional Definitions

[0067] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0068] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0069] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary’ skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.Atty. Dkt. No. 650053.01247

[0070] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0071] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0072] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone. A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ’ B or “A and B.”

[0073] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0074] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to useAtty. Dkt. No. 650053.01247a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.” Illustrative embodiments1. A peptibody comprising a nematode asparaginyl-tRNA synthetase, or a fragment thereof, linked to an antibody constant region.2. The peptibody of embodiment 1 , wherein the antibody constant region comprises an Fc region.3. The peptibody of embodiment 1 or 2, wherein the nematode asparaginyl-tRNA synthetase comprises Brugia asparaginyl-tRNA synthetase.4. The peptibody of any one of the preceding embodiments, wherein the nematode asparaginyl-tRNA synthetase comprises Brugia malayi asparaginyl-tRNA synthetase.5. The peptibody of any one of the preceding embodiments, wherein the nematode asparaginyl-tRNA synthetase consists of Brugia malayi asparaginyl-tRNA synthetase.6. The peptibody of any one of the preceding embodiments, wherein the nematode asparaginyl-tRNA synthetase comprises a sequence with at least 85% identity to SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 with at least 85% identity’ to SEQ ID NO: 1.7. The peptibody of any one of the preceding embodiments, wherein the nematode asparaginyl-tRNA synthetase comprises an N-terminal fragment of Brugia asparaginyl- tRNA synthetase.8. The peptibody of any one of the preceding embodiments, wherein the nematode asparaginyl-tRNA synthetase comprises SEQ ID NO: 2 or a sequence with at least 85% identity to SEQ ID NO: 2.9. The peptibody of any one of the preceding embodiments, wherein the antibody constant region comprises SEQ ID NO: 3, or a sequence with at least 85% identity to SEQ ID NO: 3.10. The peptibody of any one of the preceding embodiments, wherein the peptibody is a one-armed peptibody, wherein the one-armed peptibody comprises a first subunit and a second subunit, wherein the first subunit comprises the asparaginyl-tRNA synthetase linked to an antibody Fc region and the second subunit comprises an antibody Fc region, and wherein the first subunit and the second subunit are linked together.11. The peptibody of any one of embodiments 1-10, wherein the peptibody is an inverse agonist of CXCR1 or CXCR2.Atty. Dkt. No. 650053.0124712. A polynucleotide comprising a sequence encoding the peptibody of any one of embodiments 1-11.13. The polynucleotide of embodiment 12, wherein the polynucleotide further comprises a regulatory sequence operably linked to the sequence encoding the peptibody. 14. The polynucleotide of embodiment 13, wherein the regulator ■ sequence comprises an enhancer or a promoter.15. A cell comprising the polynucleotide of any one of embodiments 12-14.16. The cell of embodiment 15, wherein the cell is a mammalian cell.17. The cell of embodiment 16, wherein the cell is a HEK 293 cell, a CHO cell, or a NIH323 cell.18. A method comprising administering a therapeutically effective amount of the peptibody of any one of embodiments 1-11 to a subject in need thereof.19. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the peptibody of any one of embodiments 1-11 to a subject in need thereof.20. A method of making a peptibody, the method comprising cultivating the cell of any one of embodiments 15-17 and enriching, isolating, or purifying the peptibody.21. A pharmaceutical composition comprising the peptibody of any one of embodiments 1-11.EXAMPLES

[0075] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Example 1 - Brugia malayi asparaginyl-tRNA synthetase facilitates immune evasion by functioning as an inverse agonist and blocks the normal signal transduction pathway mediated by CXCL8 receptors

[0076] Chemokines and cancer biology are intertwined subjects. CXCL8 (interleukin-8, IL-8) is a proinflammatory chemokine that interacts with G protein coupled receptors (GPCRs), CXCR1 and CXCR2, on a wide variety of cells, and plays a key role in tumor biology, cancer pathogenesis, tumor angiogenesis and survival, and response to chemotherapy [1-5], CXCL8 has been proposed as a biomarker of tumor growth. [3-arrestins are multifunctional intracellular proteins with an ability to directly interact with GPCRs. 0-arrestins contribute to multiple aspects of GPCR signaling, trafficking, and downregulation and, thus, like GPCRs, fyarrestins have emerged as important targets in new cancer therapies [6-8], Melanoma, lymphoma, prostate, lung,Atty. Dkt. No. 650053.01247pancreatic, neural, head neck, breast and ovarian cancers all exhibit abnormal CXCL8 expression and therefore clinical trials have explored candidate CXCR1 / 2 antagonists as cancer treatments [9-14], CXCL8 inhibition is under study in patients receiving immune checkpoint inhibitors to enhance anti-tumor activity. CXCL8 can activate multiple signaling pathways by direct GPCR interaction with or without P-arrestin-mediated signaling, and it is not easy to predict which one or more pathways will be involved with any new ligand. Inhibitors may elicit different activities on different signaling cascades, a phenomenon known as ligand-biased signaling.

[0077] A new and emerging concept in human immunology is that certain eukaryotic aminoacyl-tRNA synthetases (AARS) have one function when intracellular, and other unpredicted functions when secreted extracellularly where they impact a wide array of physiological processes. Extra-cellular AARS with new extracellular functions are known as “physiocrines” [15-20],

[0078] BmAsnRS (Brugia malayi asparaginyl-tRNA synthetase) is the first example of a parasite-derived physiocrine. The inventors’ prior work revealed that the BmAsnRS exhibits at least four activities: aminoacylation of asparagine, ATPase activity7, synthesis of diadenosine oligophosphates, and a novel immunological activity7via CXCL8 chemokine receptors [21-27] . See Ramirez, Zack Howard and Kron (2006). Brugia malayi asparaginyl-transfer RNA synthetase induces chemotaxis of human leukocytes and activates G-protein-coupled receptors CXCR1 and CXCR2. J Infec Dis., which is incorporated by reference herein in its entirety. Regarding the immunological activity, the inventors published all of the following original observations: BmAsnRS is the most highly expressed AARS in Brugia malayi, 10 times more highly expressed than the other 19 other cytoplasmic AARS; secreted BmAsnRS, chemoattracts human and murine cells that express CXCL8 receptors, CXCR1 and CXCR2; chemoattraction by BmAsnRS is associated with induction of MAP kinase phosphory lation as in CXCL8 signaling, but BmAsnRS did not induce a calcium transient upon binding as does CXCL8. Pretreatment of cells with BmAsnRS blocks the calcium transient of CXCL8; BmAsnRS interacts with the second extracellular receptor loop of CXCR1 and CXCR2 in chimeric cell lines, whereas CXCL8 binds to different extracellular loops; In the NFKB signal transduction pathway of human immature dendritic cells. BmAsnRS and CXCL8 exhibit opposite patterns of gene expression; the inventors solved the atomic structure of BmAsnRS to confirm it had the same modular structure as other class 2 eukaryotic AARS, and learned that the amino terminus folds to produce significant three-dimensional overlap with CXCL8 and at least one other CXC chemokine, SDF-1 (CXCL12); only the N terminus of BmAsnRS interacts with CXCR1 / 2 by using differentAtty. Dkt. No. 650053.01247extracellular loops compared to CXCL8; the N terminus is attached via a 33-residue unstructured linker to a high molecular weight domain that yields a 63 kDa protein approximately 60 times the mass of CXCL8. The mass of monoclonal antibodies and peptibodies are approximately 60kDa and have the potential to alter GPCR receptor internalization kinetics; intraperitoneal treatment of T cell transfer mice dying of colitis (100% lethal) completely reverses colonic inflammation and results in 100% survival

[0028] ,

[0079] Until recently the inventors did not understand fully how BmAsnRS and CXCL8 were causing opposite responses via the same receptors. New data presented here uses highly sensitive GPCR G protein dissociation assays to compare the effects of equimolar CXCL8 and BmAsnRS using transiently transfected HEK cells expressing CXCR1 and CXCR2 [Figure 1].GPCR G protein dissociation assays measure bioluminesence resonance energy transfer (BRET) to quantify the amounts of G protein subunit proteins that are associated (basal state) and dissociated (internalized). CXCL8 exhibits a concentration dependent decrease in associated G protein, consistent with the activation of the ligand / receptor complex. BmAsnRS blocks the CXCL8 response, however in the absence of CXCL8, BmAsnRS increases the amount of associated G protein, a feature of inverse agonists. To put these results in perspective for cancer chemotherapy, small molecule or monoclonal antibody antagonists of CXCL8 are in clinical trials but very few inverse agonists have been described.

[0080] This work draws parallels between parasites and cancers as masters of immune evasion, and parasite-derived physiocrines as novel anti-cancer therapeutics because CXCL8 pathways are important in the pathogenesis of many cancers. This disclosure represents as innovative approach to study CXCL8 inhibition using a parasite physiocrine that evolved a GPCR-specific mechanism to evade host immune responses - inverse agonism. Introduction of new methods such as GPCR dissociation assays to study ligand-receptor interactions may provide new insight into existing CXCL8 '■antagonists" already commercially available.

[0081] The inventors will test the hypothesis that BmAsnRS is a novel inverse agonist of CXCR1 / 2 and that novel activity can be recreated in a minimally antigenic form via peptibody technology that incorporates an unusual CXCR1 / 2 binding domain with a high molecular carboxy¬ terminus in which IgGFc is substituted for the large carboxy terminus of BmAsnRS. The structure of BmAsnRS [FIG. 2] demonstrates a classical 3-part modular form characteristic of class II AARS in which the ATP binding domain (antiparallel beta sheets) resides in in the large carboxy terminus 111-548 residues. A detached amino terminus is an evolutionarily new domain not present in prokary otic AARS and it interacts with CXCR1 / 2 because the residues fold to create aAtty. Dkt. No. 650053.01247topology that binds to CXC8 chemokine receptors in an unusual way. Chemoattraction by BmAsnRS is mediated only by the N terminus [FIG. 3], Because the amino acid sequence of CXCL8 and BmAsnRS demonstrate minimal homology, the inventors expressed each domain of the BmAsnRS and tested each for chemoattraction of neutrophils. Only the amino terminus induces chemotaxis. It is known also that the N terminus of human AsnRS (CCR3), human histidyl-tRNA synthetase (CCR5) and Schistosoma japonicum AsnRS (monocytes) all elicit chemoattraction but do so via specific and different chemokine receptors. None of these other 3 physiocrines interact with CXCL8 receptors [29,30].

[0082] Significant structural homology exists between BmAsnRS, CXCL8 and CXCL12 (SDF-1). To better understand why BmAsnRS induced chemoattraction, the inventors solved the complete atomic structure of BmAsnRS and demonstrated its three-component modular structure typical of class 2 eukaryotic AARS [FIG.2], Next, the inventors compared the three-dimensional structure of BmAsnRS N terminus with chemokine structures available in the NCBI database. Computational analysis of the N-terminal structure using DaliLite (ekhidna.biocenter.helsinki.fi / dali_lite / start) identified a region that overlaid with the chemokine SDF-1 (Stromal Cell Derived Factor- 1 / CXCL 12). consisting of the [3 hairpin-a helix motif common to chemokines FIG. 4], The same structural fold found in CXCL8 is shown overlaid on the N-terminal region in BmAsnRS and human SDF-1. DaliLite superimposed the backbone of residues Lys A36-Lys A45, Lys A48-Ser A55, and Lys A56-Gln A68 of BmAsnRS onto residues Ala A35-Asn A44, Asn A45-Asp A52, and Leu A55-Asn A67 of SDF-1 (PDB entry 2k03), with a Ca RMSD of 2.3 A .The LIRTKKDGKQ(V / I)W amino acid sequence of the [3 hairpin in the BmAsnRS chemokine motif is similar to that of other CXC chemokines, matching all but the final position:,I,L)(I,V.A)(A.V,R)(T,S,K,W.L)(L,K.M)(K,N,S)(N,D)(O,K,N)(G,O) (R,K,E,Q,S,V)(K,Q,E,I) (V,I,L) (C).

[0083] The secreted parasite physiocrine, BmAsnRS, facilitates immune evasion by functioning as an inverse agonist and blocks the normal signal transduction pathway mediated by CXCL8 receptors. The inventors conclude that BmAsnRS is an inverse agonist because of two structural features: (1) the receptor binding domain of BmAsnRS (N terminus) binds to different extracellular receptor loops compared to CXCL8, and (2) the N terminus of BmAsnRS is linked to a high molecular weight carboxy terminal domain, resulting in the native BmAsnRS proteinAtty. Dkt. No. 650053.01247approximately the same mass as a monoclonal antibody or peptibody. Therefore, a prototype therapeutic synthetic peptibody that possesses both key structural features of BmAsnRS, and is of the same molecular weight as BmAsnRS, may be a novel therapeutic immunomodulatory agent in CXCL8 responsive cancers. Given the importance of CXCL8 and [3-arrestins in cancer biology, and the NCI challenge to identify “Drugs from Bugs”, the inventors’ prior work justifies a closer study of BmAsnRS as a novel inverse agonist for CXCR1 / 2.

[0084] Determine if BmAsnRS activation of CXCR1 / 2 is associated with recruitment of P arrestin pathways.

[0085] P-arrestins function in desensitization of GPCRs, control of GPCR intracellular trafficking and activation of multiple signaling pathways and are useful in discover}' of ligands for orphan GPCRs [6,7]. Therefore, study of BmAsnRS in P-arrestin recruitment assays will provide important new data to understand signal transduction. The inventors anticipate P-arrestin recruitment assays will redemonstrate the dose-response relationships the inventors observed in G protein dissociation assays. Furthermore, given structural homology between the N terminus of BmAsnRS and SDF-1 (CXCL12), P-arrestin recruitment assays may identify GPCRs other than CXCR1, CXCR2 and CXCL12 that interact with BmAsnRS. Knowledge of unexpected receptor activations may be important to understand therapeutic potential.

[0086] TANGO high throughput screening platform identifies interactions with expected GPCRs and orphan / unpredicted receptors [FIG. 5,

[0031] ]. The PRESTO TANGO platform was developed to facilitate the rapid, efficacious, parallel and simultaneous profiling of biologically active compounds across the entire human druggable GPCRome. Advantages of this assay in high-throughput screening include its independence from GPCR coupling, high signal-to-background ratios and amplification of small initial inputs into large readout signals Biased signaling or functional selectivity of pathways involves cell signaling through multiple pathways.

[0087] Experimental Approach

[0088] 1. P arrestin recruitment assays using CXCR1 and CXCR2 cells. Upon activation of the GPCR by a ligand, P-arrestin fusion protein is recruited to the C terminus of the receptor. This is followed by cleavage of the GPCR fusion protein at the TEV protease site. Cleavage results in release of the rTA transcription factor, which after transport to the nucleus, activates transposition of the luciferase reporter gene. Modular design of TANGO constructs is shown in FIG.5.

[0089] 2. P arrestin recruitment assays using full library of non-olfactory GPCRs if P arrestins are involved with BmAsnRS binding. High throughput screening using the TANGO-P-arrestin method is an ideal way to simultaneously screen all (~ 300) non-olfactory GPCRs forAtty. Dkt. No. 650053.01247interaction with BmAsnRS or its peptibodies. The inventors anticipate at least CXCR1, CXCR2 and the SDF-1 receptor, CXCR12, will be identified. The opportunity to screen the libraries available will maximize the inventors' ability to detect unpredicted or unexpected activation of GPCRs.

[0090] Interpretation of results. Log drug concentration is plotted along the X axis and Bret Ratio of GPCR dissociation along the Y axis. Each point represents the mean of quadruplicate replicates. Curves are fitted using GraphPad Prism software. Dose response relationships determine IC50 values. Controls for each assay will include known ligands (BmAsnRS, commercially available CXCR1 / 2 antagonists, as well as a purified BmAsnRS protein into which the inventors have introduced point mutations at key residues in the amino terminus of BmAsnRS that abolish chemoattraction

[0024] ,

[0091] Statistical analysis. Data points are the mean + / - SEM of typical experiments done in quadruplicate and curves are fitted using GraphPad Prism 5.0.

[0092] Specific Aim 2. To determine effects of a human IgG Fc-BmAsnRS peptibody on GPCRs

[0093] Hypothesis. An inverse agonist peptibody based on the structure of BmAsnRS requires two features: [1] a non-traditional domain that binds to CXCL8 receptors, i.e. the BmAsnRS N terminus (1-111) and [2] a high molecular weight domain (437 aa) bound to the 1-111 domain, that yields the same overall mass as native BmAsnRS (63 kDa).

[0094] Rationale. Peptibody technology is an elegant way to study protein-protein interactions

[0034] , Whereas a molecule with the same molecular weight as CXCL8 could function as an agonist or antagonist at the receptor, inverse agonist activity is a novel observation not previously reported for a parasite molecule, and rarely reported for commercially available CXCL8 receptors.

[0095] Experimental Appr^^Ai.

[0096] 1. Application of peptibody technology to mimic activity of BmAsnRS. A therapeutic peptibody is constructed from two recombinant Fc fusion proteins that dimerize to form the final molecule [FIG. 6] T inventors already have designed, produced and purified plasmids and proteins that express: N terminus alone (13 kDa), Fc alone (25 kDa), one armed peptibody (63 kDa) and 2 armed peptibody (76 kDa) that is composed of two 38 kDa proteins. Because the inventors intend to study the activity of peptibodies in CXCL8-responsive cell lines and animal models, the Fc domain in these proteins is engineered with mutations to minimize complement- and antibody-mediated toxicities. To determine if the novel N terminus ofAtty. Dkt. No. 650053.01247BmAsnRS is required for inverse agonist activity in a peptibody, the inventors will express a control peptibody that displays BmAsnRS residues 12-548 on human IgGFc. The inventors will synthesize another control peptibody that displays native CXCL8 as a one-armed peptibody. Lastly, another peptibody control will be constructed as a two-armed peptibody in which both Fc fragments display the 1-111 domain. Comparative studies of one-armed vs. two-armed peptibodies may assess if two 1-111 domains interfere with receptor binding due to steric hindrance.

[0097] 2. G protein dissociation assays. Concentration-response curves for each peptibody are determined in parallel with BmAsnRS, CXCL8 and other controls. Peptibodies exhibiting G protein dissociation curves similar to native BmAsnRS will be retested in P-arrestin assays using (a) CXCR1 and CXCR2, and (b) the inventors’ full GPCR library of 300+ if BmAsnRS binding is found to involve -arrestins.

[0098] It is known that some assays are better than others for demonstration of inverse agonism. Because some labs believe that cAMP second messenger assays provide the best overall measurement of inverse agonism in cellular assays, any peptibody the inventors generate that shows inverse agonism in p-arrestin recruitment or GPCR dissociation assays, will be studied in cAMP assays. Many commercially available small molecule and monoclonal antibody CXCR1 / 2 antagonists have not been studied in GPCR dissociation assays. Thus, the inventors are well aware that the choice of an assay is crucial for determination of pharmacological effects and this will guide the inventors’ thinking and interpretation of all results. The inventors plan to obtain several commercially available CXCR1 / 2 antagonists to compare their activity to lead peptibodies: Ladarixin, a dual CXCR1 / 2 antagonist; SX-682 an orally bioavailable antagonist that binds to CXCR1 and CXCR2 to inhibit their activation by tumor-secreted chemokines; SB22502,a nonpeptide selective inhibitor of CXCR2 that can inhibit CXCL8 and GRO-a-mediated Ca2+ mobilization; Reparixin, another CXCR 1 / 2 antagonist. [35-38]

[0099] Specific Aim 3: To determine effects of BmAsnRS / peptibody on human cancer cells.

[0100] Hypothesis. BmAsnRS and selected peptibodies with inverse agonist activity' at CXCR1 / 2 will disrupt colon cancer and melanoma cell CXCR1 / 2 internalization kinetics, cell viability, proliferation and resistance to chemotherapy. Effects on cancer cells may not be directly cytotoxic but will significantly impair their functional capacities to survive or metastasize. BmAsnRS is not directly cytotoxic to human and murine leukocytes.Atty. Dkt. No. 650053.01247

[0101] Rationale. If the cancer cells express CXCR1 / 2 receptors (or new orphan receptors yet unidentified), they will be impacted by BmAsnRS peptibodies. However, the potency of inverse agonism may differ depending on the density of CXCR1 / 2 expressed on the surface of specific cell lines.

[0102] Experimental Approach. CXCL8 responsive melanoma cell lines (A375SM and TXM-13) and colon cancer cells (LoVo, SW620) are grown in culture as previously reported [4,H]. Effects of BmAsnRS, peptibody, CXCL8 and controls (commercially available CXCR1 / 2-antagonists specified herein) are measured using various methods to determine cell viability, proliferation, receptor internalization and cancer drug resistance per standard protocols [39-41], RNA will be extracted from all cells and stored at -80° C for future use to examine gene expression using pathway specific- and total microarrays as previously described

[0028] ,

[0103] Interpretation of Results and Statistical analysis. Receptor internalization is measured before and after incubation with BmAsnRS, peptibody, CXCL8 and positive / negative controls. Surface receptor expression measurement is performed by labeling with antibody against extracellular domains. All assays are run in quadruplicate and p values <0.05 via t-tests are considered significant.

[0104] Potential pitfalls and alternative approaches. Several assays exist for measuring surface expression of GPCRs

[0033] . Three classes of tests to assess cancer drug resistance are found in the literature, i.e. fresh tumor cell culture tests, cancer biomarker tests and positron emission tomography (PET) tests

[0041] , The inventors anticipate that BmAsnRS and peptibody will inhibit the behavior or function of cancer cell lines in vitro. Peptibodies with maximum activity will be identified for future work studying the in vivo effects of BmAsnRS / peptibody using murine xenograft models. All the cancer cell lines specified in this grant are in use in the inventors’ Cancer Center Network of laboratories. Thus, quality control measures are routinely done to confirm receptor expression and genetic lineages. Standard biohazard protocols are in place.

[0105] From prior work the inventors know that native 63 kDa BmAsnRS is not cytotoxic to human and murine leukocytes but can dramatically affect downstream gene expression and disrupt inflammatory pathways. Also, the inventors know that in persons naturally infected with Brugia malayi secreted BmAsnRS is antigenic but the antibodies do not neutralize the biological activity of BmAsnRS and are not associated with any discernable pathology [unpublished.] It is possible inverse agonist peptibodies could affect chemokine receptors other than CXCR1 / 2 on cancer cell lines. The inventors do anticipate that receptors for CXCL12 will interact withAtty. Dkt. No. 650053.01247peptibody inverse agonists because the inventors have already shown conformational overlap between CXCL12 and BmAsnRS [FIG. 4], Similarly, if the inventors identify through TANGO screening that other GPCRs are affected by BmAsnRS, then the inventors predict that the diversity of biological activities mediated by an inverse agonist peptibody immunomodulator could be considerable [42-45] .

[0106] Literature cited1. Ha H, Debnath B and Neamati N (2017). Role of the CXCL8-CXCR1 / 2 axis in Cancer and Inflammatory Disease. Theranostics (7) 6: 1543-88. PMID 28529637.2. Alfaro C, Sanmamed M. Rodriguez-Ruiz M, et al. (2017). Interleukin-8 in cancer pathogeneses, treatment and followup. Cancer Treatment Reviews 60:24-31. PMID 28866366.3. Hughes CE, Nibbs RJ (2018). A guide to chemokines and their receptors. The FEBS Journal 285; 2944-2971. PMID 29637711.4. Ning J. Mangegold PC, Hong KY, et al (2011). Interleukin 8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models. Int J Cancer 128(9): 2038-2049. PMID 206485595. Xiong X. Liao X, Qiu S. et al (2022). CXCL8 in Tumor Biology and its implications for Clinical Translation. Front Mol Biosci 9:723-846. PMID 35372515.6. Wang T, Li Z, Cvjic ME, Krause C, Zhang L. Sum CS (2017). Measurement of Beta Arrestin Recruitment for GPCR Targets. In Assay Guidance Manual, Bethesda.Markossian, Grossman and Brimacombe, et al, editors. PMID 29165975.7. Violin JD, Lefkowitz RJ (2007). Beta Arrestin biased ligands at seven transmembrane receptors. Trends in Pharm Sciences 28:8; 416-422. PMID 17644195.8. Hutchings C. Cseke G, Osborne G, Woolard J, et al (2013). Monoclonal anti -Beta 1 adrenergic receptor antibodies activate G protein signaling in the absence of beta-arrestin recruitment. Monoclonal antibodies 6:1, 246-261. PMID 24253107.9. Shukla A K. Dwivedi-Agnihortri (2020). GPCR Signaling in cancer. Advances in Cancer Research 145: 1-156. PMID 32089163.10. Singh JK. Simoes BM, Howell SJ et al (2013). Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells. Breast Cancer Research 5:210. 1-9. PMID 24041156.11. Huang S, Mills L, Mian B, et al (2002). Fully Humanized Neutralizing Antibodies to Interleukin-8 (ABX-IL*) Inhibit Angiogenesis, Tumor Growth and Metastasis of Human Melanoma. Am J Pathology 161 (1):125-134. PMID 12107097.12. Qazi BS, Tange K and Qazi A (2011). Recent Advances in Underlying Pathologies provide insight into interlukin-8 expression-mediated inflammation and angiogenesis.International Journal of Inflammation: 1-13. PMID 22235381.Atty. Dkt. No. 650053.0124713. Chan LP, Liu C, Chiang FY, et al (2017). IL-8 promotes inflammatory mediators and stimulates activation of p38 MAPK / ERK-NF-kB pathway and reduction of JNK in HNSCC. Oncotarget. vol 8(34); 56375-56388. PMID 218915597.14. Korbecki J, KupnickaP et al (2022). CXCR2 receptor: regulation of expression, signal transduction and involvement in cancer. Int J Molec Sciences 23, 2168. PMID 35216283.15. Lo WS, Gardiner E. Xu Z, et al (2014). Human tRNA synthetase Catalytic Nulls with Diverse Functions. Science July 18;345(6194):328-332. PMID: 25035493.16. Yakobov N, Debard S, Fischer F, et al. (2018). Cytosolic aminoacyl-tRNA synthetases: Unanticipated relocation for unexpected functions. BBA Gene Regulatory Mechanisms 1861:387-400. PMID 29155070.17. Tzima E, Schimmel P (2006). Inhibition of tumor angiogenesis by a natural fragment of atRNA synthetase. Trends Biochem Sci 31:7-10. PMID 16297628.18. Wakasugi K, Schimmel P (1999). Two distinct cytokines released from a human aminoacyl-tRNA synthetase. Science. 284(5411):147-51. PMID 10102815.19. Kron M, Haertlein M (2005). Aminoacyl-tRNA synthetases and Disease, in The Aminoacyl-tRNA Synthetases, ed. M Ibba, C Francklyn, S Cusack, Landes Bioscience.20. Guo M, Yang X-L, Schimmel P (2010). New functions of tRNA synthetases beyond translation. Nat. Rev. Mol. Cell. Biol. 11:668-674. PMID 20700144.21. Kron M, Marquard K, Hartlein M, Price S, Leberman R. (1995). An immunodominant antigen of Brugia malayi is an asparaginyl-tRNA synthetase. FEBS Lett. Oct 23 ;374(1): 122-4. PMID: 7589498.22. Ramirez BL, Howard OM, Dong HF, Edamatsu T, Gao P, Hartlein M, Kron M. Brugia malayi asparaginyl-transfer RNA synthetase induces chemotaxis of human leukocytes and activates G-protein-coupled receptors CXCR1 and CXCR2. J Infect Dis.2006 Apr 15;193(8): 1164-71. PMID: 16544258.23. Kron M, Leykam J, Kopaczewski J, Matus I. Identification of diadenosine triphosphate in Brugia malayi by reverse phase high performance liquid chromatography and MALDI mass spectrometry' . J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Sep l;856(l-2):234-8. doi: 10.1016 / j.jchromb.2007.06.014. Epub2007 Jun 23. PMID: 17631429; PMCID: PMC2044565.24. Chandrasekar R, Sivanesan S, Natarajan M, Naveena K, Preetha N, Karthika S, Vimalraj S, Kron M, Dhanasekaran A (2021). Evaluation of the angiogenic properties of Brugia malayi asparaginyl-tRNA synthetase and its mutants: A study on the molecular target for antifilarial drug development. Mol Biochem Parasitol. Nov;246:l 11426.PMID:34666104.25. Crepin T, Peterson F, Haertlein M, Jensen D, Wang C, Cusack S, Kron M (2012). A hybrid structural model of the complete Brugia malayi cytoplasmic asparaginyl-tRNA synthetase. J Mol Biol. Jan 28 ;405 (4): 1056-69. PMID: 21134380.Atty. Dkt. No. 650053.0124726. Kron MA, Wang C, Vodanovic-Jankovic S. Howard OM, Kuhn LA (2012).Interleukin-8-like activity in a filarial asparaginyl-tRNA synthetase. Mol Biochem Parasitol. Sep;185(l):66-9. PMID:22710390.27. Kron M, Petridis M, Milev Y, Leykam J, Haertlein M (2003). Expression, localization, and alternate function of asparaginyl-tRNA synthetase in Brugia malayi. Mol Biochem Parasitoll29;33-39. PMID 12798504.28. Kron M, Metwali A, Jancovic S, Elliott D (2013). Nematode AsnRS resolves submucosal inflammation in T- cell transfer colitis mice. Clinical and Vaccine Immunology 20(2):276-281. PMID: 23254300.29. Peck Y. Pickering D. Mobli M, Kron M, Loukas A, Daly N (2023). Solution structure of the N-terminal extension domain of a Schistosoma japonicum asparaginyl-tRNA synthetase. J Biomol Struct Dyn 12:1-11. PMID: 37572327.30. Howard OM, Dong HF, Yang D, Raben N, Nagaraju K, Rosen A, Casciola-Rosen L, Hartl ein M, Kron M, Yang D, Yiadom K, Dwivedi S, Plotz PH, Oppenheim JJ (2002). Histidyl-tRNA synthetase and asparaginyl-tRNA synthetase, autoantigens in myositis, activate chemokine receptors on T lymphocytes and immature dendritic cells. J Exp Med 196, 781-791. PMID 12235211.31. Kroeze W, Sassano M, Huang X, Lansu K, McCorvy J, Giguere P, Sciaky N, Roth B (2015). PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat Struct Mol Biol 22(5):362-9. PMID 25895059.32. Zhou Y, Meng J, et al (2021). Multiple GPCR Functional Assays Based on Resonance Energy Transfer Sensors. Frontiers in Cell Developmental Biology 9;1-16. PMID 34041234.33. Avet C, Mancini A, Breton B, et al (2022). Effector membrane translation biosensors reveal G protein and Parrestin coupling profiles of 100 therapeutically relevant GPCRs. eLife 11 ;e74101 PMID 35302493.34. Cavaco M , Castanho MA, Neves V (2017). Peptibodies: An elegant solution for a long-standing problem. Biopolymers 21. PMID: 29266205.35. Landoni G, Zangrillo A, Piersanti G, Scquizzato T, Piemonti L (2022). The effect of reparixin on survival in patients at high risk for in-hospital mortal ity : a meta-analysis of randomized trials. Front Immunol. Jul 25;13:932251. PMID: 35958623.36. Li E, Yang X, Du Y, Wang G, Chan DW, Wu D, Xu P, Ni P, Xu D, Hu Y (2021). CXCL8 Associated Dendritic Cell Activation Marker Expression and Recruitment as Indicators of Favorable Outcomes in Colorectal Cancer. Front Immunol. Mav 7;12:667177. PMID: 34025668.37. Citro A, Valle A, Cantarelli E, Mercalli A, Pellegrini S, Liberati D, Daffonchio L, Kastsiuchenka O, Ruffini PA, Battaglia M, Allegretti M, Piemonti L (2015). CXCR1 / 2 inhibition blocks and reverses type 1 diabetes in mice. Diabetes 64(4): 1329-40. PMID: 25315007.Atty. Dkt. No. 650053.0124738. Chapman RW, Minnicozzi M, Celly CS, et al (2007). A novel, orally active CXCR1 / 2 receptor antagonist, Sch527123, inhibits neutrophil recruitment, mucus production, and goblet cell hyperplasia in animal models of pulmonary inflammation. J Pharmacol Exp Ther 322(2):486-93. PMID: 17496165.39.Beerepoot P, Lam VM, Salahpour A (2013). Measurement of G protein-coupled receptor surface expression. J Recept Signal Transduct Res 33(3): 162-165. PMID 23557016.40.Yu Z, Vodanovic-Jancovic S, LedeboerN, Kron M (2011). Tirandamycins from Streptomvces sp 179944 Inhibiting the parasite Brugia malayi asparagine tRNA synthetase. Org Lett 15; 13(8):2034-2037. PMID 21405052.41. Lippert T, Ruoff H, Volm M. Current Status of Methods to Assess Cancer Drug Resistance (2011). Int J Med Sci 8(3):245-253. PMID 21487568.42. Vilardaga JP, Steinmeyer R, Harms G.S and Lohse MJ (2005). Molecular basis of inverse agonism in a G-protein coupled receptor. Nat Chem Biol 1,25-28.PMID: 16407989.43 Barreda-Gomez G, Giralt MT, Rodriguez-Puertas R (2010). Methods to measure G- protein coupled receptor activity for the identification of inverse agonists. Methods Enzymology 485:261- 273. PMID: 21050922.44 Sum CS, Murphy BJ, Li Z, Wang T, Zhang L, Cvijic ME (2019). Pharmacological Characterization of GPCR Agonists, Antagonists, Allosteric Modulators and Biased Ligands from HTS Hits to Lead Optimization. In: Markossian S, Grossman A, Brimacombe K, Arkin M, Auld D, Austin C, Baell J, Chunget al, . Assay Guidance Manual [Internet], Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences. PMID: 31693331.45 . Qin J, Cai Y, Xu Z, et al (2022). Molecular mechanism of agonism and inverse agonism in the ghrelin receptor. Nature Communications 13:300. PMID 35027551.Example 2 - treatment of a subject suffering from a disease or disorder associated with CXCL8 expression using the disclosed peptibodies

[0107] In one example, a subject suffering from a disease or disorder associated with CXCL8 expression, e.g., cancer, is administered an amount of a composition comprising the disclosed peptibodies. The peptibodies may suitably be administered by any route that is indicated by the particular treatment needs of the subject, e.g., oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. Signs and symptoms of the disease or disorder may be reduced by the administration of the peptibody. Treatment may be administered daily, weekly, monthly, yearly, or on a schedule as determined by the patient's progress, pursuant to a physician's decision. It is anticipated that the subject will experience a reduction in tumor size, overall tumor burden, reduction in tumor number, reductionAtty. Dkt. No. 650053.01247in tumor grade, remission, or cure of the disease or disorder, or other metrics associated with reduction in signs or symptoms of a disease or disorder associated with CXCL8 expression, as compared to an untreated subject. Methods of measuring reductions in signs and symptoms of diseases associated with CXCL8 expression, e.g., cancer, are known in the art.

[0108] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0109] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.SequencesAtty. Dkt. No. 650053.01247

Claims

Atty. Dkt. No. 650053.01247CLAIMS1. A peptibody comprising a nematode asparaginyl-tRNA synthetase, or a fragment thereof, linked to an antibody constant region.

2. The peptibody of claim 1, wherein the antibody constant region comprises an Fc region.

3. The peptibody of claim 1 or 2, wherein the nematode asparaginyl-tRNA synthetase comprises Brugia malayi asparaginyl-tRNA synthetase.

4. The peptibody of claim 3, wherein the nematode asparaginyl-tRNA synthetase consists of Brugia malayi asparaginyl-tRNA synthetase.

5. The peptibody of claim 3, wherein the nematode asparaginyl-tRNA synthetase comprises SEQ ID NO: 1 or a sequence with at least 85% identify to SEQ ID NO:

1.

6. The peptibody of claim 1, wherein the nematode asparaginyl-tRNA synthetase comprises an N-terminal fragment of Brugia malayi asparaginyl-tRNA synthetase.

7. The peptibody of claim 6, wherein the N-terminal fragment of the nematode asparaginyl-tRNA synthetase comprises SEQ ID NO: 2 or a sequence with at least 85% identity to SEQ ID NO: 2.

8. The peptibody of claim 1, wherein the antibody constant region comprises SEQ ID NO: 3, or a sequence with at least 85% identify to SEQ ID NO: 3.

9. The peptibody of claim 1, wherein the peptibody is a one-armed peptibody, wherein the one-armed peptibody comprises a first subunit and a second subunit, wherein the first subunit comprises the asparaginyl-tRNA synthetase linked to an antibody Fc region and the second subunit comprises an antibody Fc region, and wherein the first subunit and the second subunit are linked together.

10. The peptibody of claim 1, wherein the peptibody is an inverse agonist of CXCR1 or CXCR2.

11. A polynucleotide comprising a sequence encoding the peptibody of claim 1.Atty. Dkt. No. 650053.0124712. The polynucleotide of claim 1, wherein the polynucleotide further comprises a regulator^' sequence operably linked to the sequence encoding the peptibody.

13. The polynucleotide of claim 12, wherein the regulator}7sequence comprises an enhancer or a promoter.

14. A cell comprising the polynucleotide of any one of claims 11-13.

15. The cell of claim 14, wherein the cell is a mammalian cell.

16. The cell of claim 15, wherein the cell is a HEK 293 cell, a CHO cell, or an NIH323 cell.

17. A method comprising administering the peptibody of claim 1 to a subject.

18. A method of treating a disease or disorder associated with CXCL8 expression in a subject in need thereof, the method comprising administering the peptibody of claim 1 to a subject in need thereof.

19. The method of claim 18, wherein the disease or disorder associated with CXCL8 expression comprises cancer.

20. The method of claim 19, wherein the cancer is selected from the group consisting of melanoma, lymphoma, prostate, lung, pancreatic, neural, head and neck, breast, and ovarian cancers.

21. A method of making a peptibody. the method comprising cultivating the cell of claim 14 and enriching, isolating, or purifying the peptibody.

22. A pharmaceutical composition comprising the peptibody of claim 1 and a pharmaceutically acceptable excipient.

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