WO2025030147A1

WO2025030147A1 - Extracellular vesicles for inhibiting interleukin 6 (il6 trans-signaling and vascular epidermal growth factor (vegf)-signaling

Info

Publication number: WO2025030147A1
Application number: PCT/US2024/040820
Authority: WO
Inventors: Rob HALENBECK; Andrew Carr; Maria E. FUENTES; Jennifa Gosling; Colin GOTTLIEB; Alex MOK
Original assignee: Mantra Bio, Inc.
Priority date: 2023-08-02
Filing date: 2024-08-02
Publication date: 2025-02-06

Abstract

Disclosed herein are engineered extracellular vesicles for inhibiting 1L6 trans-signaling and VEGF- signaling comprising (a) an IL6/IL6 receptor alpha complex-binding extracellular vesicle (EV) protein comprising a GP130 domain 1-3 protein portion, for binding IL6/1LR. receptor alpha complex, and a chimeric vesicle localization moiety, and (b) a VEGF-binding EV protein comprising at least two VEGFR- domain fusion units, for binding VEGF, and a chimeric vesicle localization moiety, wherein each VEGFR-domain fusion unit comprises an Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) linked to an Ig-like domain 3 of VEGF receptor 2 (VEGFR-2), and wherein the chimeric vesicle localization moiety comprises: (i) a surface-and-transmembrane domain of a first vesicle localization moiety, and (ii) a cytosolic domain of a second vesicle localization moiety, the method of making said vesicle and uses thereof,

Description

EXTRACELLULAR VESICLES FOR INHIBITING INTERLEUKIN 6 (IL6) TRANS- SIGNALING AND VASCULAR EPIDERMAL GROWTH FACTOR (VEGF)-SIGNALING

[ 1 ] All references, including publications, patent applications, patents, and accession numbers from publicly available databases cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[2] This application contains a Sequence Listing that has been filed electronically in the form of a XML filed computer-readable nucleotide/ amino acid sequence listing submitted concurrently herewith and identified as follows: the text file named "SEQ IL6", which was created on August 1, 2024, the entirety of which is hereby incorporated by reference.

FIELD OF THE DISCLOSURE

[3] The present disclosure relates, in general, to extracellular vesicles for inhibiting interleukin 6 (IL6) trans-signaling and vascular epidermal growth factor (VEGF)-signaling and uses thereof.

BACKGROUND

[4] Diabetic retinopathy (DR) is a major complication of diabetes mellitus and it is the major cause of visual loss in the working-age population (Wang et al., Int. J. Molec. Sci., 2018, 19: 1816; Lechner et al., Vision Res., 2017, 139: 7-14). Diagnosis is made based on clinical manifestations of retinal abnormalities. DR can be divided into non-proliferative (NPDR) and proliferative disease (PDR). NPDR is the early stage of the disease, where there are manifestations of inflammation, and capillary occlusion. At this early stage, patients can be asymptomatic but display pathologies like microaneurysms, hemorrhages and exudates, which can be detected by fundus angiography. PDR is the most advanced form of the disease, and it is characterized by neovascularization accompanied by severe visual impairment. The main cause of the visual impairment is the development of diabetic macular edema (DME), which is characterized by thickening of the macula due to liquid accumulation caused by breakdown of the blood-retinal barrier. Importantly, DME can occur at any stage of the disease.

[5] The first line of treatment for DR/DME is the use of agents that block the vascular endothelial growth factor (VEGF) pathway. The aim of this treatment is to decrease angiogenesis and vascular leakage and improve visual acuity. However, significant improvement in visual acuity is achieved only in a fraction of the patients. In addition, in a significant proportion of patients there is persistent DME (Brown et al., Ophthalmol., 2013, 120:2013-22; Gonzalez et al., Am. J. Ophthalmol., 2016, 172:72-79; Bressler et al., JAMA Ophthalmol., 2018, 136:257-269). This partial or lack of response in a significant number of patients is likely to be the contribution of other pathways to the disease pathogenesis. One of those pathways that has been identified to significantly contribute to the disease is the IL-6 pathway. Inflammation is an important component of the pathogenesis of DR and DME. Chronic inflammation has been described in both patients and animal models of the disease. Significant high levels of IL-6 have been detected in ocular fluids of patients, and its levels correlate with disease severity (Chen et al., J. Ophthalmol., 2023, 2023:3987281. doi: 10.1155/2023/3987281 ; Chen et al., Molec. Vis., 2016, 22:1005).

[6] In addition, an important role for IL-6 in the disruption of both endothelial and epithelial barriers have been described, as well as a role of IL-6 in activation of endothelial cells and angiogenesis (Valle et al., Exp. Eye Res., 2019, 178:27; Droho et al., Sci. Rep., 2021, 11 : 18084; Izumi-Nagai et al., Am. J. Pathol. , 2007, 170(6):2149-58).

[7] For that reason, the use of the combination of antagonists of the VEGF and IL-6 pathways could result in significant improvement of efficacy in patients that don’t respond well to VEGF pathway inhibitors alone. Other diseases where the combination of inhibiting activity of IL-6 and VEGF pathways could be beneficial are retinal vein occlusion, dry eye, dry and wet age-related macular degeneration (AMD), and retinopathy of prematurity.

[8] Current anti-VEGF therapies are lacking in full efficacy ignoring significant contribution of other pathways, such as IL6, to disease pathophysiology. No single active agent exists that inhibits both VEGF-signaling and IL6-trans-signaling pathways. This current void in a single therapeutic agent targeting both pathways is filled by an engineered extracellular vesicle (EV) that inhibits both VEGF-signaling and IL6 trans-signaling pathways. The use of such a therapeutic agent could lead to significantly better outcomes in the treatment of DR/DME and pathophysiological conditions and diseases associated with excess VEGF-signaling and IL6 trans- signaling. SUMMARY OF THE INVENTION

[9] Provided herein are extracellular vesicles (e.g., exosomes) for inhibiting interleukin 6

(IL6) trans-signaling and vascular epidermal growth factor (VEGF)-signaling comprising: (a) an IL6/IL6 receptor alpha complex-binding extracellular vesicle (EV) protein comprising a GP130 domain 1-3 protein portion, for binding IL6/IL6 receptor alpha complex, and a vesicle localization moiety (e.g., a chimeric vesicle localization moiety), and (b) a VEGF-binding EV protein comprising at least two VEGFR-domain fusion units, for binding VEGF, and a chimeric vesicle localization moiety. Each VEGFR-domain fusion unit comprises an Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) linked to an Ig-like domain 3 of VEGF receptor 2 (VEGFR-2). Further, the chimeric vesicle localization moieties comprise: (i) a surface-and-transmembrane domain of a first vesicle localization moiety, and (ii) a cytosolic domain of a second vesicle localization moiety. The invention also provides compositions containing a plurality of extracellular vesicles (e.g., exosomes) of the invention wherein about 75% of the extracellular vesicles and/or exosomes are mature or processed proteins that recognize and bind both the IL6/IL6 receptor alpha complex and VEGF, e.g., both mature or processed MB02 and mature or processed MB010 proteins. The invention further provides nucleic acids, vectors, methods of making and using the EVs or exosomes.

[10] The EV for inhibiting IL6 trans-signaling and VEGF-signaling provided herein can allow selective binding to soluble IL6/IL6R complex and soluble VEGF, and thereby, simultaneously sequester soluble IL6/IL6R complex and soluble VEGF, preventing binding to gpl30 cO-receptor and VEGF receptor on cell surfaces, respectively. By sequestering the soluble IL6/IL6R complex, the EVs (e.g,, exosomes) of the invention inhibit IL6 trans-signaling associated with inflammation, oxidative stress, and endothelial barrier disruption in human retinal endothelial cells. By sequestering VEGF, the EVs and/or exosomes of the invention inhibit VEGF-signaling associated with neo-angiogenesis/neovascularization and vascular permeability.

ADVANTAGES OF THE INVENTION

[11] The use of EVs of the invention provides several distinct advantages. In general, the

EV has been engineered to comprise a high concentration of IL6/IL6R-binding and VEGF-binding proteins, so as to have a high capacity for sequestering soluble IL6/IL6R complex and soluble VEGF, and hence, a high potency for inhibiting both IL6/IL6R trans-signaling and VEGF-signaling. The presence of hundreds of binding partners for IL6/IL6R complex or VEGF on the surface of an EV provides superior avidity. By specifically binding IL6/IL6R complex but not IL6 or IL6R alone, the EV and/or exosome of the invention specifically inhibits IL6 trans-signaling without inhibiting classic 1L6 signaling, thereby inhibiting or reducing inflammation, oxidative stress, and endothelial barrier disruption without immune-suppressive effects. When used to treat eye disease or conditions, such as DR and/or DME, the EV of the invention has enhanced therapeutic half-life, minimizing drug clearance and increasing durability by localizing to the vitreous humor and retina but not to the aqueous humor, following delivery by intravitreal injection.

BRIEF DESCRIPTION OF FIGURES OF THE INVENTION.

[12] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[13] Figure 1: Vesicle flow cytometry was used to determine abundance of gp!30 (1A- C) or chimeric VEGFR (1D-E) on extracellular vesicles (EVs).

[14] Figure 2: Inhibition of VEGF signaling (2A-B) or IL6 trans-signaling (2C-E) in HUVEC cells.

[15] Figure 3: Monospecific (A) and bispccific (B) exosomes were assessed for their ability to inhibit VEGF ligand activity using the VEGFR2 phosphorylation assay in HUVEC cells.

[16] Figure 4: Abundance of gpl30 (PE-A) and VEGFR (APC-A) on EVs determined by vesicle flow cytometry.

[17] Figure 5: Biodistribution of TEV following single injection in rabbit eye.

[18] Figure 6: Lesion area in rat 15 days post laser induced CNV measured by fluorescein angiography.

[19] Figure 7: Effects of TEV on pSTAT3 induction in mouse endotoxin induced uveitis model.

[20] Figure 8: Exemplary plasmid map or vector for expression of the IL6/IL6 receptor alpha-binding EV protein and VEGF-binding protein with MB04 protein expression vector as an example.

[21] Figure 9: Schematic of representative VEGF-binding EV proteins anchored to a surface of an extracellular vesicle . [22] Figure 10: Schematic of representative IL6/IL6 receptor alpha-binding EV proteins anchored to a surface of an extracellular vesicle .

DETAILED DESCRIPTION OF THE INVENTION

[23] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described. References to exemplary nucleic acid and amino acid sequences and, when applicable their respective SEQ ID NOs, are provided in the Tables herein.

Extracellular Vesicles of the Invention

[24] The invention provides extracellular vesicles for inhibiting interleukin 6 (IL6) trans- signaling and vascular epidermal growth factor (VEGF)-signaling comprising: (a) an IL6/IL6 receptor alpha complex-binding extracellular vesicle (EV) protein comprising a GP130 domain 1-3 protein portion, for binding IL6/IL6 receptor alpha complex, and a vesicle localization moiety (e.g., a chimeric vesicle localization moiety (chimeric VLM)), and (b) a VEGF-binding EV protein comprising at least two VEGFR-domain fusion units, for binding VEGF, and a vesicle localization moiety (e.g., a chimeric VLM). Each VEGFR-domain fusion unit comprises an Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) linked to an Ig-like domain 3 of VEGF receptor 2 (VEGFR-2), and wherein the vesicle localization moiety comprises: (i) a surface-and-transmembrane domain of a first vesicle localization moiety, and (ii) a cytosolic domain of a second vesicle localization moiety. In an embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein further comprises GP130 domain 4, 4-5, or 4-6, such that the GP130 domain 1-3 protein portion is extended in the carboxyl direction resulting in a GP130 domain 1-4, 1 -5, or 1-6 protein portion, respectively, as would be found in portions of a full length GP130 protein.

[25] The GP130 domain

[26] For example, the GP130 may be a mammalian GP130. Examples of mammalian GP130 include, but are not limited to, a primate, camel, horse, dog, fox, tiger, lion, lynx, squirrel, cheetah, cat, rat, mouse, hamster, deer, elephant, rabbit, cow, sheep, goat, pig, panda, bear, bat, seal, walrus, dolphin, porpoise, and whale GP130. Examples of primates include any of a monkey, gorilla, chimpanzee, gibbon, orangutan, lemur, macaque, marmoset, panda, baboon and human. [27] In one embodiment, the mammalian GP130 may be a human GP130, its homologue from a mammal, or a variant with at least 85% sequence identity to human GP130 domain 1-3, domain 1-4, domain 1-5, or domain 1-6 protein portion. Merely by way of example, a full length human GP130 comprises an amino acid sequence as provided in NCBI Reference Sequence:

NP_002175.2, or UniProtKB Accession number: P40189-1 , (both incorporated by reference) or an amino acid sequence:

MPKSYLPQTVRQGGYMPQ (SEQ ID NO: XXI (also referred to herein as SEQ ID NO: 40)).

[28] In another embodiment, the GP130 domain 1-3 protein portion comprises an amino acid sequence starting from glutamic acid at position 23 to aspartic acid at position 324 of NCBI

Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1 (both incorporated by reference) or an amino acid sequence of:

ED (SEQ ID NO: XX2 (also referred to herein as SEQ ID NO: 41)).

[29] In a further embodiment, the GP130 domain 1-4 protein portion comprises an amino acid sequence starting from glutamic acid at position 23 to histidine at position 425 of NCBI

Q ( Q ( referred to herein as SEQ ID NO: 42)).

[30] In yet a further embodiment, the GP130 domain 1-5 protein portion comprises an amino acid sequence starting from glutamic acid at position 23 to alanine at position 517 of NCBI

Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1, (both incorporated by reference) or an amino acid sequence of:

GPGS PES IKAYLKQA (SEQ ID NO: XX4 (also referred to herein as SEQ ID NO: 43)).

[31] In an additional embodiment, the GP130 domain 1-6 protein portion comprises an amino acid sequence starting from glutamic acid at position 23 to glutamic acid at position 619 of

NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1 , (both incorporated by reference) or an amino acid sequence of:

Q Q

XX5 (also referred to herein as SEQ ID NO: 44)).

[32] In accordance with the invention, the homologue of a mammal or variant thereof

(with at least 85% sequence identity to human GP 130) is a corresponding homologous GP130 portion from any of amino acid sequence having NCBI Reference Sequence Accession Number or GenBank Accession Number of KAI2537527.1, AAI17403.1, XP_030859139.1, XP 032003087.1, XP 003827430.1, XP_055108393.1, XP 001144416.2, XP 030653699.1, XP_024102831.1, KAI4021315.1, XP_024102830.1, XP_030859138.1, XP_054343962.1, XP 054343961.1, EAW54938.1, KAI2537532.1 , XP 050651531 .1 , XP_010384765.1, XP_045249658.1, NP_001252920.1, XP 023066294.1, EHH54264.1, XP 007971047.1, XP 011852135.1 , XP_014995508.1, XP_045249657.1, XP 033035485.1, XP 024102832.1, XP 011945381.1, XP 054343964.1, XP_012312716.1 , XP_017404172.1, NP_001254695.1, XP 035133154.1, XP_054102576.1, XP 003925890.1, XP 037866327.1, XP 011945382.1, XP 008568260.1, XP_010964581.1 , XP_010976061.1, XP_040308910.1, XP_011281197.1, XP_006206072.1 , XP 020932176.1 , XP 047696155.1 , XP_004416362.1 , XP_032260293.1 , XP 025748119.1, XP 049505435.1, XP_020041462.1, XP 007079992.1, NP 001273950.1, XP_055187683.1, XP 045349502.1 , XP 045750990.1, XP 021557427.1, XP_055187685.1, VFV38139.1, XP 038513637.1, XP_046942608.1, XP_019269821.1, XP_047614529.1, XP 035569431.2, XP 030176981.1, XP 011232783.1, XP_034870332.1, EFB 18858. l, NP_001090901.1 , XP 025874618.1, XP 035921574.1, XP_004744695.1, XP_047585592.1 , XP_045650281.1, XP 038513638.1 , XP 008689627.1 , XP 041621151.1 , XP 044080554.1 , XP 055187686.1 , XP_032706291.1, XP 032192101 .1 , XP_036701589.1, XP_012510757. 1 , XP_007195663.1 , XP_041621153.1, XP_029798115.1, XP_029072211.1, KAI5774350.1, XP_004422884.1, XP_020932174.1, XP 045855414.1, XP_039100444.1, XP_004275204.1 , XP 030699355.1, and

XP 024621 122.1.

[33] In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may have at least 90% sequence identity to the human GP130 portion. In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may have at least 95% sequence identity to the human GP130 portion. In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may have at least 98% sequence identity to the human GP130 portion.

[34] In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may comprise at least 85% sequence identity to the human GP130 portion and optionally one or more amino acid insertion, so long as the insertion does not affect binding of the GP130 portion to IL6/IL6 receptor alpha complex. In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may comprise at least 90% sequence identity to the human GP130 portion and optionally one or more amino acid insertion, so long as the insertion does not affect binding of the GP130 portion to IL6/IL6 receptor alpha complex. In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may comprise at least 95% sequence identity to the human GP130 portion and optionally one or more amino acid insertion, so long as the insertion does not affect binding of the GP130 portion to IL6/IL6 receptor alpha complex. In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may comprise at least 98% sequence identity to the human GP130 portion and optionally one or more amino acid insertion, so long as the insertion does not affect binding of the GP130 portion to IL6/IL6 receptor alpha complex.

[35] In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may comprise at least 85% sequence identity to the human GP130 portion and optionally one or more amino acid deletion, so long as the deletion does not affect binding of the GP130 portion to IL6/IL6 receptor alpha complex. In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may comprise at least 90% sequence identity to the human GP130 portion and optionally one or more amino acid deletion, so long as the deletion does not affect binding of the GP130 portion to IL6/IL6 receptor alpha complex. In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may comprise at least 95% sequence identity to the human GP130 portion and optionally one or more amino acid deletion, so long as the deletion does not affect binding of the GP130 portion to IL6/IL6 receptor alpha complex. In an embodiment, the GP130 portion of the homologue of a mammal or variant thereof may comprise at least 98% sequence identity to the human GP130 portion and optionally one or more amino acid deletion, so long as the deletion does not affect binding of the GP130 portion to IL6/IL6 receptor alpha complex.

[36] In one embodiment, GP130 domain 1 comprises a conserved glutamine-asparagine dipeptide found within 150 amino acids from N-terminus of GP130 or comprises threonine, arginine, or glutamine eleven amino acids upstream of the conserved glutamine-asparagine dipeptide. In an embodiment, the threonine is located eleven amino acids upstream of the conserved glutamine- asparagine dipeptide within 150 amino acids from N-terminus of GP130. In an embodiment, the threonine is threonine- 102 eleven amino acids upstream of the conserved glutamine- 113/asparagine- 114 dipeptide of human GP130 as provided in NCBI Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1 (incorporated by reference) or threonine-77 eleven amino acids upstream of glutamine-88/asparagine-89 of SEQ ID NO: XX6 (also referred to herein as SEQ ID NO: 45).

[37] Additionally, in an embodiment of the invention, the conserved glutamine-asparagine dipeptide found within 150 amino acids from N-terminus of GP130 and the threonine, arginine, or glutamine eleven amino acids upstream of the conserved glutamine-asparagine dipeptide may be mutated at a single residue, both residues or al! three residues, to increase the binding affinity of the 1L6/IL6 receptor alpha complex-binding EV protein for IL6/IL6 receptor alpha complex. In accordance with the invention, the threonine, arginine, or glutamine may be mutated to a tyrosine and the glutamine-asparagine dipeptide may be mutated, replacing glutamine with phenylalanine and/or replacing asparagine with leucine, singly or in combination, so as to increase binding affinity of the IL6/IL6 receptor alpha complex-binding EV protein for IL6/IL6 receptor alpha complex. In an embodiment, the threonine eleven amino acids upstream of the glutamine-asparagine dipeptide and the glutamine and asparagine residues of the dipeptide are mutated to tyrosine, phenylalanine, and leucine, either singly or in combination, so as to increase binding affinity of the IL6/IL6 receptor alpha complex-binding EV protein for IL6/IL6 receptor alpha complex. In an embodiment, the threonine- 102, glutamine- 113, and asparagine- 114 of human GP130 as provided in NCBI Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1 (both incorporated by reference) or threonine-77, glutamine-88, and asparagine-89 of SEQ ID NO: XX6 (also referred to herein as SEQ ID NO: 45) are mutated singly or in combination to tyrosine, phenylalanine, and leucine, respectively, so as to increase binding affinity of the IL6/IL6 receptor alpha complex- binding EV protein for IL6/IL6 receptor alpha complex. In an embodiment, the threonine- 102, glutamine- 113, and asparagine- 114 of human GP130 as provided in NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1 (both incorporated by reference) or threonine-77, glutamine-88, and asparagine-89 of SEQ ID NO: XX6 (also referred to herein as SEQ ID NO: 45) are mutated to tyrosine, phenylalanine, and leucine, respectively, so as to increase binding affinity of the IL6/IL6 receptor alpha complex-binding EV protein for IL6/IL6 receptor alpha complex. In this embodiment, homologous GFP130 portion or variant of human GP130 portion corresponding to threonine- 102, glutamine- 113, and asparagine- 114 of human GP130 as provided in NCBI Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1 (both incorporated by reference) or threonine-77, glutamine-88, and asparagine-89 of SEQ ID NO: XX6 (also referred to herein as SEQ ID NO: 45) are mutated to tyrosine, phenylalanine, and leucine, respectively, so as to increase binding affinity of the IL6/IL6 receptor alpha complex- binding EV protein for IL6/IL6 receptor alpha complex.

[38] In an embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein GP130 portion with increased binding affinity for IL6/IL6 receptor alpha complex comprises a threonine, arginine, or glutamine eleven amino acid residues upstream of a phenylalanine- asparagine dipeptide. In an embodiment of the invention, the IL6/IL6 receptor alpha complex- binding EV protein GP130 portion with increased binding affinity for IL6/IL6 receptor alpha complex comprises an arginine, or glutamine eleven amino acid residues upstream of a glutamine- leucine dipeptide. In an embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein GP130 portion with increased binding affinity for IL6/IL6 receptor alpha complex comprises a tyrosine eleven amino acid residues upstream of a glutamine-asparagine dipeptide or a phenylalanine-asparaginc dipeptide. In an embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein GP130 portion with increased binding affinity for IL6/IL6 receptor alpha complex comprises a tyrosine eleven amino acid residues upstream of a glutamine-leucine dipeptide. In an embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein GP130 portion with increased binding affinity for IL6/IL6 receptor alpha complex comprises a threonine, arginine, or glutamine eleven amino acid residues upstream of a phenylalanine-leucine dipeptide. In a preferred embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein GP130 portion with increased binding affinity for IL6/IL6 receptor alpha complex comprises a tyrosine eleven amino acid residues upstream of a phenylalanine-leucine dipeptide.

[39] In an embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein with increased binding affinity for IL6/IL6 receptor alpha complex comprises a mammalian GP130 protein portion with homology to human GP130 domain 1 comprising T/R/Q102Y, QI 13F or N 1 14L substitution, or a combination thereof, based on amino acid sequences corresponding to human GP130 sequence as provided in NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1 (both incorporated by reference). In a preferred embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein with increased binding affinity for IL6/IL6 receptor alpha complex comprises a mammalian GP130 protein portion with homology to human GP130 domain 1 comprising T/R/Q102Y, QI 13F or N114L substitution, or a combination thereof, based on amino acid sequences corresponding to human GP130 sequence as provided in NCBI Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1 (both incorporated by reference).

[40] In a more preferred embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein with increased binding affinity for IL6/IL6 receptor alpha complex comprises domain 1 of human GP130 portion with T102Y, QI 13F or N114L substitution, or a combination thereof, based on human GP130 sequence as provided in NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1 (both incorporated by reference). In a most preferred embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein with increased binding affinity for IL6/IL6 receptor alpha complex comprises domain 1 of human GP130 portion with T102Y, QI 13F and N1 14L substitutions based on human GP130 sequence as provided in NCBI Reference Sequence: NPJD02175.2, or UniProtKB Accession number: P40189-1 (both incorporated by reference).

[41] In a further example, the GP130 domain one (1) may comprise an amino acid sequence starting from aspartic acid at position 26 to isoleucine at position 120 of NCBI Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1, (both incorporated by reference) or an amino acid sequence of: DPCGYI SPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTI PKEQYTI INRTASSVTFTDIASLN IQLTCNILTFGQLEQNVYGITI (SEQ ID NO: XX6 (also referred to herein as SEQ ID NO: 45)), wherein the conserved glutamine-asparagine dipeptide is underlined along with the threonine eleven amino acids upstream of the conserved glutamine-asparagine dipcptidc. In this embodiment, the threonine, arginine, or glutamine upstream of the conserved glutamine-asparagine dipeptide may be threonine at amino acid position 102 and the glutamine-asparagine dipeptide is at amino acid positions 113 and 114 of NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1 (both incorporated by reference), or threonine at amino acid position 77 and glutamine-asparagine at amino acid positions 88 and 89 of SEQ ID NO: XX6 (also referred to herein as SEQ ID NO: 45). For example, the threonine-102 is mutated to tyrosine (T102Y), the glutamine- 113 of the dipeptide is mutated to phenylalanine (QI 13F), or the asparagine- 114 of the dipeptide is mutated to leucine (N114L), or a combination thereof, to increase binding affinity of the IL6/IL6 receptor alpha complex-binding EV protein for IL6/IL6 receptor alpha complex.

[42] VEGF-binding EV proteins

[43] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the VEGF-binding EV protein comprising the Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) comprises an amino acid sequence from serine at position 129 to isoleucine at position 230 of NCBI Reference Sequence Accession Number: NP_002010.2 or UniProtKB Accession Number: Pl 7948-1, (both incorporated by reference) or an amino acid sequence of:

[44] WDS

[45]

referred to herein as SEQ ID NO: 86)), or an amino acid sequence having 90% sequence identity thereof.

[46] In some embodiments, the VEGF-binding EV protein comprising an Ig-like domain 3 of a VEGF receptor 2 (VEGFR-2) comprises an amino acid sequence from aspartic acid at position 225 to lysine at position 327 of NCBI Reference Sequence Accession Number: NP 002244.1 or UniProtKB Accession Number: P35968-1, (both incorporated by reference) or an amino acid sequence of:

[47]

[48]

referred to herein as SEQ ID NO: 87)), or an amino acid sequence having 90% sequence identity thereof.

[49] In additional embodiments, the VEGF-binding EV protein comprising the VEGFR- domain fusion unit comprises an amino acid sequence of:

AS SGLMTKKNST FVRVHEK (SEQ ID NO: XX47 (also referred to herein as SEQ ID NO: 88)), or an amino acid sequence having 90% sequence identity thereof. [51] In further embodiments, the amino acid sequence having 90% sequence identity to the Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) is selected from a corresponding homologous VEGFR-1 portion from any of amino acid sequence having NCBI Reference Sequence Accession Number or GcnBank Accession Number of XP 055100552.1, XP_034792394.1,

XP 008969852.1, XP_032006460.1 , XP 032006459.1, XP_055100550.1, XP_003818375.1, PNJ48512.1 , XP_032006458.1, PNJ48513.1, XP O55100549.1, AYL88758.1, XP_025220259.1, XP 009189959.1, XP 008020010.1, XP_015294597.1, XP_028693312.1 , XP_025220258.1, XP 015294596.1, XP 030674446.1, XP 003270267.1, EHH58499.1, XP_025220257.1, XP 01 1940270.1 , XP 003913768.1 , XP _005585612.1 , XP_008020007.1 , XP_001117928.3, El 11128920.1 , XP-003270266.1 , XP_033092342.1 , XP 023064515.1, XP_011753897.1 , XP 017703974.1 , XP_021531864.1 , XP 015294597.2, XP 033092333.1 , XP 033092324.1 , XP_023064514.1, XP 015294596.2, XP 017703973.1, XP_032150285.1, XP 011753896.1 , XP 023064513.1, XP 033092313.1, XP 005585612.2, XP_017703972.1, XP 021531863.1, XP 032150284.1, XP_010360667.2, XP_010360657.2, XP_030778324.1, XP 017392985.1, XP_010360648.2, XP_011814493.1 , and XP 017392983.1.

[52] Additionally, in some embodiments, the amino acid sequence having 90% sequence identity to the Ig-like domain 3 of a VEGF receptor 2 (VEGFR-2) is selected from a corresponding homologous VEGFR-2 portion from any of amino acid sequence having NCBI Reference Sequence Accession Number or GenBank Accession Number of BAD93138.1 , AAI31823.1 ,

XP 01 1708980.1 , XP.,007996878.1 , XPJ05555327.1, XP_014994176.1 , EHH26004.1, XP 031520318.1 , EHH53800.1 , XP_01 1928915.1 , XPJ)11847889.1, XP 011708979.1 , XP 005555327.2, XP 011928917.1, XP_025240971.1, XP 003268434.2, XP 032130119.1, XP 012296575.1, XP 002745866.2, XP 032130121.1, XP 012296576.1, XP 032130122.1 , XP 054109329.1, XP 008991591.1, XP_032130120.1, XP_055150622.1, XP_032007207.1, XP 007121194.1, XP_017395743.1, XP_028347631.1, XP_028347630.1, XP_028347633.1, XP_003933665.1, XP_017395745.1, XP_028347632.1, XP_017395746.1, XP 017395744.1 , XP 010361178.1, XP_011817095.1, XP_023048010.1, XP_036709011.1, XP_019783716.1, X P 030736423.1 , XP_022419698.1 , XP_029090747.1 , XP_00426831 1.1 , XP JJ30615937.1 , KAJ8795398.1 , XP 036709012.1, XP 007168490.2, TKC45787.1 , XP_033281298.1, XP_022419718.1, XP 033281299.1, XP_022419727.1, XP_030736425.1, XP_030736424.1, XP_019783717.1, XP 019783718.1, XP 042792296.1, XP_033281300.1, XP_019325208.1, XP_047712387.1, XP_049486434.1, XP_042792297.1, XP_043428557.1, XP_042840444.1, XP 040339255.1, XP 026912793.1, XP Ol 1280181.3, VFV35517.1, XP_030169713.1,

XP 046947373.1, XP_007075942.2, XPJM7712388.1, XP_049486433.1, XP 040339249.1, XP_033070538.1, XP_025777172.1, KAB1282375.1, XP_006177399.1, and XP_031290309.1.

[53] In yet further embodiments, the amino acid sequence having 90% sequence identity to the VEGFR-domain fusion unit is selected from the group of sequences homologous to:

[54] (a) the Ig-like domain 2 of a VEGF receptor I (VEGFR-1) of SEQ ID NO: XX45 (also referred to herein as SEQ ID NO: 86) present in NCBI Reference Sequence Accession Number or GenBank Accession Number of XP_055100552.1, XP 034792394.1, XP 008969852.1 , XP_032006460.1, XP_032006459.1, XP_O55100550.1, XP 003818375.1, PNJ48512.1, XP_032006458.1, PNJ48513.1, XPJ)55100549.1, AYL88758.1, XP 025220259.1 ,

XP 009189959.1, XP_008020010.1, XP_015294597.1, XP 028693312.1, XP_025220258.1,

XP 015294596.1, XP 030674446.1, XP_003270267.1, EHH58499.1, XP_025220257.1,

XP 01 1940270.1 , XP_003913768.1, XP_005585612.1, XP 008020007.1, XP 001117928.3, EHH28920.1, XP_003270266.1, XP_033092342.1, XP 023064515.1, XP_011753897.1, XP 017703974.1, XP 021531864.1 , XP_015294597.2, XP 033092333.1 , XP_033092324.1 , XP 023064514.1, XP_015294596.2, XP_017703973.1, XP 032150285.1 , XP_011753896.1, XP 023064513.1, XP 033092313.1, XP_005585612.2, XP_017703972.1, XP_021531863.1 , XP 032150284.1, XP_010360667.2, XP 010360657.2, XP 030778324.1, XP_017392985.1, XP 010360648.2, XP_011814493.1 , or XP_017392983.1 , and

[55] (b) the Ig-like domain 3 of a VEGF receptor 2 (VEGFR-2) of SEQ ID NO: (also referred to herein as SEQ ID NO: 87) present in NCBI Reference Sequence Accession Number or GenBank Accession Number of B AD93138.1, AAI31823.1 , XP_011708980.1 , XP 007996878.1 , XP 005555327.1, XP_014994176.1, EHH26004.1, XP 031520318.1, EHH538OO.1,

XP 01 1928915.1 , XP 01 1847889.1 , XP 011708979.1, XP_005555327.2, XP 011928917.1 , XP_025240971.1 , XP_003268434.2, XP 032130119.1, XP_012296575.1, XP_002745866.2, XP_032130121.1, XP_012296576.1, XP 032130122.1, XP 054109329.1, XP_008991591.1, XP 032130120.1, XP 055150622.1, XP 032007207.1, XP_007121194.1, XP_017395743.1, XP 028347631.1, XP 028347630.1, XP_028347633.1, XP_003933665.1, XPJ) 17395745.1, XP 028347632.1 , XP_017395746.1, XP_017395744.1, XP_010361178. l, XP_011817095.1, XP 023048010.1, XP 036709011.1, XP 019783716.1, XP 030736423.1, XP 022419698.1, XP 029090747.1, XP 004268311 .1, XP 030615937.1, KAJ8795398.1, XP 036709012.1 , XP 007168490.2, TKC45787.1 , XP_033281298.1 , XP 022419718.1 , XP_033281299.1, XP_022419727.1, XP_030736425.1, XP_030736424.1, XP_019783717.1, XP 019783718.1 , XP 042792296.1 , XP_033281300.1, XP_019325208.1, XP_047712387.1, XP_049486434.1, XP 042792297.1, XP_043428557.1, XP_042840444.1, XP_040339255.1, XP_026912793.1 , XP_01 1280181.3, VFV35517.1, XP_030169713.1, XP_046947373.1, XP_007075942.2, XP 047712388.1, XP_049486433.1, XP 040339249.1, XP 033070538.1, XPJJ25777172.1, KAB1282375.1, XP 006177399.1, and XP_031290309.1.

[56] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the VEGF-binding EV protein additionally comprises a third constant immunoglobulin domain, CH3 domain, of IgGl heavy chain. For example, the IgGl heavy chain is human. In a specific, embodiment, the CH3 domain of human IgGl heavy chain comprises an amino acid sequence starting from glycine at position 110 to lysine at position 216 of GenBank Accession Number: AAL96263.1, or an amino acid sequence of:

[57] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDS

[58] DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: XX9 (also referred to herein as SEQ ID NO: 49)).

[59] 1'he invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the VEGF-binding EV protein additionally comprises a flexible linker. In one embodiment, the flexible linker is a glycine-serine rich linker. For example, the glycine-serine rich linker comprises greater than 80% glycine and serine residues. In another embodiment, the glycine- serine rich linker comprises 100% glycine and serine residues. Merely by way of example, the glycine-serine rich linker may comprise a linker length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. Examples of suitable glycine-serine rich linkers include, but are not limited to, any of (GS)₃ (SEQ ID NO: XXI 0 (also referred to herein as SEQ ID NO: 50), (GS)4 (SEQ ID NO: XXI 1 (also referred to herein as SEQ ID NO: 51), (GS)₅ (SEQ ID NO: XX12 (also referred to herein as SEQ ID NO: 52), (GS)e (SEQ ID NO: XXI 3 (also referred to herein as SEQ ID NO: 53), (GS)₇ (SEQ ID NO: XXI 3B (also referred to herein as SEQ ID NO: 54), (GS)_g (SEQ ID NO: XX14 (also referred to herein as SEQ ID NO: 55), (GS)₉ (SEQ ID NO: XX15 (also referred to herein as SEQ ID NO: 56), (GS)io (SEQ ID NO: XXI 6 (also referred to herein as SEQ ID NO: 57), (GGS)₂ (SEQ ID NO: XX17 (also referred to herein as SEQ ID NO: 58), (GGS)₃ (SEQ ID NO: XXI 8 (also referred to herein as SEQ ID NO: 59), (GGS)4 (SEQ ID NO: XXI 9 (also referred to herein as SEQ ID NO: 60), (GGS)₅ (SEQ ID NO: XX20 (also referred to herein as SEQ ID NO: 61), (GGS)₆ (SEQ ID NO: XX21 (also referred to herein as SEQ ID NO: 62), (GGGS)₂ (SEQ ID NO: XX22 (also referred to herein as SEQ ID NO: 63), (GGGS)₃ (SEQ ID NO: XX23 (also referred to herein as SEQ ID NO: 64), (GGGS)4 (SEQ ID NO: XX24 (also referred to herein as SEQ ID NO: 65), (GGGS)s (SEQ ID NO: XX25 (also referred to herein as SEQ ID NO: 66), (GGGGS)₂ (SEQ ID NO: XX26 (also referred to herein as SEQ ID NO: 67), (GGGGS)a (SEQ ID NO: XX27 (also referred to herein as SEQ ID NO: 68), (GGGGS)4 (SEQ ID NO: XX28 (also referred to herein as SEQ ID NO: 69), GGGGGS (SEQ ID NO: XX29 (also referred to herein as SEQ ID NO: 70), (GGGGGS)₂ (SEQ ID NO: XX30 (also referred to herein as SEQ ID NO: 71), (GGGGGS)₃ (SEQ ID NO: XX31 (also referred to herein as SEQ ID NO: 72), (SG)₂S (SEQ ID NO: XX32 (also referred to herein as SEQ ID NO: 73), (SG)₃S (SEQ ID NO: XX33 (also referred to herein as SEQ ID NO: 74), (SG)4S (SEQ ID NO: XX34 (also referred to herein as SEQ ID NO: 75), (SG)sS (SEQ ID NO: XX35 (also referred to herein as SEQ ID NO: 76), (SG)eS (SEQ ID NO: XX36 (also referred to herein as SEQ ID NO: 77), (SG)₇S (SEQ ID NO: XX37 (also referred to herein as SEQ ID NO: 78), (SG)₈S (SEQ ID NO: XX38 (also referred to herein as SEQ ID NO: 79), and (SG)gS (SEQ ID NO: XX39 (also referred to herein as SEQ ID NO: 80), or a portion thereof. In a specific embodiment, glycine-serine rich linker is any of (GS)₃ (SEQ ID NO: XXI 0 (also referred to herein as SEQ ID NO: 50), (GS)s (SEQ ID NO: XXI 2 (also referred to herein as SEQ ID NO: 52), (SG)₄S (SEQ ID NO: XX34 (also referred to herein as SEQ ID NO: 75), and (GGGGS)₃ (SEQ ID NO: XX27 (also referred to herein as SEQ ID NO: 68).

[60] Merely by way of example, the flexible linker may be located between VEGFR- domain fusion units in a head-to-tail concatemer and between an adjacent VEGFR-domain fusion unit and the chimeric vesicle localization moiety.

[61] In some embodiments, the VEGFR-domain fusion units in a head-to-tail concatemer comprises an arrangement from N-to-C-terminus: [[(VEGFR-1 domain 2)-(VEGFR-2 domain 3)]- linker]_n-[(VEGFR-l domain 2)-(VEGFR-2 domain 3)], where n = 1 for a concatemer comprising 2 VEGFR-domain fusion units, n = 2 for a concatemer comprising 3 VEGFR-domain fusion units, and n = integer z for a concatemer comprising z + 1 VEGFR-domain fusion units. For example, the linker may be any of (SG)4S, (GS)s, and (GGGGS)₃. In a specific embodiment, the linker is (SG)4S.

[62] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the VEGF-binding EV protein additionally comprises a signal peptide at its N- terminus for insertion and passage of the VEGFR-domain fusion units and the surface domain of the chimeric vesicle localization moiety through a lipid bilayer of an extracellular vesicle or an exosome. In some embodiments, the signal peptide may be present in a nascent or newly translated VEGF-binding EV protein. In some embodiments, the signal peptide may be cleaved off during or following passage of the VEGFR-domain fusion units and the surface domain of the chimeric vesicle localization moiety through the lipid bilayer of an extracellular vesicle or exosome. Merely by way of example, the signal peptide may be a mouse Ig kappa signal peptide or functional equivalence.

For example, the mouse Ig kappa signal peptide comprises an amino acid sequence from methionine at position 1 to aspartic acid at position 21 of GenBank Accession Number: AAH80787.1 , or an amino acid sequence of: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: XX40 (also referred to herein as SEQ ID NO: 81).

[63] In some embodiments, the signal peptide is absent or missing from a mature (also referred to herein as processed) VEGF-binding EV protein. In some embodiments, the extracellular vesicle or exosome comprises the processed or mature VEGF-binding EV protein. In a specific embodiment, the processed or mature VEGF-binding EV protein is found on the surface of the extracellular vesicle or exosome. Merely by way of example, the VEGFR-domain fusion units and the surface domain of the chimeric vesicle localization moiety may be found/located on the outer surface or external to the extracellular vesicle or exosome, the transmembrane domain of the chimeric vesicle localization moiety may be found/located in the lipid bilayer of the extracellular vesicle or exosome, and the cytosolic domain of the chimeric vesicle localization moiety may be found/located in the inner or interior portion of the extracellular vesicle or exosome.

[64] In some embodiment, the VEGF-binding EV protein additionally comprises an epitope tag. Examples of suitable epitope tags include, but are not limited to, a FLAG tag, a myc tag, a human influenza hemagglutinin (HA) tag, and a (His)6 tag. In a specific embodiment, the FLAG tag comprises an amino acid sequence of DYKDDDDK (SEQ ID NO: XX41 (also referred to herein as SEQ ID NO: 82). In another specific embodiment, the myc tag comprises an amino acid sequence of EQKLISEEDL (SEQ ID NO: XX42 (also referred to herein as SEQ ID NO: 83). In a further embodiment, the HA tag comprises an amino acid sequence of YPYDVPDYA (SEQ ID NO: XX43 (also referred to herein as SEQ ID NO: 84). In yet a further specific embodiment, the (His)6 tag comprises an amino acid sequence of HHHHHH (SEQ ID NO: XX44 (also referred to herein as SEQ ID NO: 85).

[65] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the VEGF-binding EV protein additionally comprises the first or second vesicle localization moiety selected from the group consisting of ACE, ADAMI 0, ADAMI 5, ADAM9, AGRN, ALCAM, ANPEP, ANTXR2, ATP1A1, ATP1B3, BSG, BTN2A1, CALM1, CANX, CD151, CD19, CD1A, CD1B, CD1C, CD2, CD200, CD200R1, CD226, CD247, CD274, CD276, CD33, CD34, CD36, CD37, CD3E, CD40, CD40LG, CD44, CD47, CD53, CD58, CD63, CD81, CD82, CD84, CD86, CD9, CHMP1A, CHMP1B, CHMP2A, CHMP3, CHMP4A, CHMP4B, CHMP5, CHMP6, CLSTN1, C0L6A1, CR1, CSF1R, CXCR4, DDOST, DELI, DLL4, DSG1, EMB, ENG, EVI2B, FUR, FASN, FCER1G, FCGR2C, FLOT1, FLOT2, FLT3, FN1, GAPDH, GLG1, GRIA2, GRIA3, GYPA, HSPG2, ICAM1, ICAM2, ICAM3, IGSF8, IL1RAP, IL3RA, IL5RA, IST1, ITGA2, ITGA2B, ITGA3, ITGA4, ITGA5, ITGA6, ITGAL, ITGAM, ITGAV, ITGAX, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, JAG1, JAG2, KIT, LAMP2, LGALS3BP, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LMAN2, LRRC25, LY75, M6PR, MFGE8, MMP14, MPL, MRC1, MVB12B, NECTIN1, N0M01, NOTCFI1, N0TCH2, NOTCH3, NOTCH4, NPTN, NRP1, PDCD1, PDCD1LG2, PDCD6IP, PDGFRB, PEC AMI , PLXNB2, PLXND1, PROMI, PTGES2, PTGFRN, PTPRA, PTPRC, PTPRJ, PTPRO, RPN1, SDC1, SDC2, SDC3, SDC4, SDCBP, SDCBP2, SELPLG, SIGLEC7, SIGLEC9, SIRPA, SLIT2, SNF8, SPN, STX3, TACSTD2, TFRC, TLR2, TMED10, TNFRSF8, TRAC, TSG101, TSPAN14, TSPAN7, . TSPAN8, TYROBP, VPS25, VPS28, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS4A,

VPS4B, VTI1A and VTI1B, or a variant or homologue thereof, and wherein the selected first and second vesicle localization moieties are different proteins and are not isoforms, or orthologs. In some embodiments, the variant is an allelic variant or an isoform. In further embodiments, the homologue is an ortholog or paralog.

[66] In specific embodiments, the surface-and-transmembrane domain of the first vesicle localization moiety of the VEGF-binding EV protein is a surface-and-transmembrane domain of LAMP2 or a variant or homologue thereof. Merely by way of example, the LAMP2 is LAMP2b or a variant or homologue thereof.

[67] In some embodiments, the cytosolic domain of the second vesicle localization moiety of the VEGF-binding EV protein is the cytosolic domain is any of PTGFRN, ITGA3, IL3RA, SELPLG, ITGB1, CLSTN1, and a variant or homologue thereof. In a specific embodiment, the cytosolic domain of the second vesicle localization moiety of the VEGF-binding EV protein is PTGFRN or a variant or homologue thereof. In another specific embodiment, the chimeric vesicle localization moiety of the VEGF-binding EV protein comprises the surface-and-transmembrane domain of LAMP2b and the cytosolic domain of PTGFRN. In accordance with the practice of the invention, the first and second vesicle localization moieties of the VEGF-binding EV protein may be human or humanized.

[68] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the surface-and -transmembrane domain of LAMP2b of the VEGF-binding EV protein comprises an amino acid sequence starting from leucine at position 29 to alanine at position 395 ofNCBI Reference Sequence Accession number NP_054701.1 , or an amino acid sequence of:

[69]

[70]

[71]

[72]

[73]

[74]

[75] I ivi VIA (SEQ ID NO: XX7 (also referred to herein as SEQ ID NO: 46).

[76] In some embodiments, the cytosolic domain of PTGFRN of the VEGF-binding EV protein comprises an amino acid sequence starting from serine at position 854 to aspartic acid at position 879 ofNCBI Reference Sequence Accession number NP 065173.2, or an amino acid sequence of: SSHWCCKKEVQETRRERRRLMSMEMD (SEQ ID NO: XX8 (also referred to herein as SEQ ID NO: 47).

[77] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the chimeric vesicle localization moiety of the VEGF-binding EV protein comprises the surface-and-transmembrane domain of LAMP2b and the cytosolic domain of PTGFRN comprising an amino acid sequence of

D (SEQ ID NO: XX8B (also referred to herein as SEQ ID NO: 48). In some embodiments, the two or more VEGFR-domain fusion units or the VEGFR-domain fusion unit concatemer are linked to the chimeric vesicle localization moiety through a flexible linker. Merely by way of example, the linker may be a glycine-serine rich linker. For example, the glycine-serine rich linker may be (GGGGS)3. In some embodiments, the chimeric vesicle localization moiety comprises the surface-and- transmembrane domain of LAMP2b and the cytosolic domain of PTGFRN.

[79] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the nascent or newly translated VEGF-binding EV protein from amino-to- carboxyl direction is organized as: N terminus-signal peptide-VEGFR-domain fusion units-linker- human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome.

[80] The some embodiments the VEGF-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-VEGFR-domain fusion units-linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, (mature or processed VEGF-binding EV protein), following incorporation into an extracellular vesicle or exosome.

[81] In some embodiments, the VEGF-binding EV protein comprises an amino acid sequence of a mature or processed MB 14 from amino acid number 22 to end of SEQ ID NO: 14 or a mature or processed MB25 from amino acid 22 to end of SEQ ID NO: 26. In some embodiment the two or more VEGFR-domain fusion units or the VEGFR-domain fusion unit concatemer are linked to the chimeric vesicle localization moiety through a peptide comprising from N-to-C direction a 1^st flexible linker, the CH3 domain, and a 2^nd flexible linker. For example, the first flexible linker and second flexible linker may be glycine-serine rich linkers. In a specific example, the first flexible linker is (86)48 (SEQ ID NO: XX34 (also referred to herein as SEQ ID NO: 75). In another specific example, the second flexible linker is (68)5 (SEQ ID NO: XX 12 (also referred to herein as SEQ ID NO; 52).

[82] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the nascent or newly translated VEGF-binding EV protein from amino-to- carboxyl direction is organized as: N terminus-signal peptide- VEGFR-domain fusion units-first linker-CH3 domain-second linker-human Lamp2b extracellular domain-and-transmembrane domain- cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome. In some embodiments, the VEGF-binding EV protein from amino- to-carboxyl direction is organized as: N terminus- VEGFR-domain fusion units-first linker-CH3 domain-second linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus (mature or processed VEGF-binding EV protein), following incorporation into an extracellular vesicle or exosome. [83] In some embodiments, the nascent or newly translated VEGF-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-signal peptidc-VEGFR-domain fusion units-first linker-CH3 domain-second linker-human Lamp2b extracellular domain-and- transmembrane domain-cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome, wherein an epitope tag may be present optionally at either N-terminus between the signal peptide and the first VEGFR-domain fusion unit or at C -terminus after the cytosolic domain of human PTGFRN.

[84] In further embodiments, the VEGF-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-VEGFR-domain fusion units-first linker-CH3 domain-second linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus (mature or processed VEGF-binding EV protein), following incorporation into an extracellular vesicle or exosome, wherein an epitope tag may be present optionally at either N- terminus before the first VEGFR-domain fusion unit or at C-terminus after the cytosolic domain of human PTGFRN.

[85] In a specific embodiment, the VEGF-binding EV protein comprises an amino acid sequence of a mature or processed MB 10 from amino acid position 22 to the end of SEQ ID NO: 10 or a mature or processed MB12 from amino acid position 22 to the end of SEQ ID NO: XX12).

[86] Vesicle Localization Moiety (VLM)

[87] In an embodiment of the invention, the GP130 portions for binding IL6/IL6 receptor alpha complex (above) and the VEGFR-domain fusion units for binding soluble VEGF (below) are incorporated into an EV and/or exosome and displayed on the outer surface of an EV and/or exosome by attaching separately to a vesicle localization moiety. In one embodiment, the GP130 portions or VEGFR-domain fusion units are attached N-terminal to the vesicle localization moiety. To facilitate passage through a lipid bilayer, a signal peptide is attached N-terminal to the GFP130 portions or VEGFR-domain fusion units. Following expression of a fusion protein comprising a signal peptide, GP130 portions or VEGFR-domain fusion units, and a vesicle localization moiety in an EV and/or exosome producing cell, the signal peptide is cleaved off from the nascent or newly translated protein following incorporation into an EV and/or exosome. In an embodiment, the EVE and/or exosome comprises a mature or processed fusion protein comprising the GP130 portions or VEGFR-domain fusion units, the vesicle localization moiety and free of the signal peptide. - [88] In a specific embodiment, the vesicle localization moiety may be a type I transmembrane protein found on the surface of an EV and/or exosome. In a preferred embodiment, the vesicle localization moiety may be a chimeric vesicle localization moiety comprising a surface- and-transmembrane domain of a first vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety, shown to synergistically increase incorporation into EVs and exosomes beyond either parental vesicle localization moiety. A complete description of such chimeric vesicle localization moieties may be found in WO2021/154888 (PCT/2021/015334, Mantra Bio Inc.), which is incorporated by reference herein.

[89] In one embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein additionally comprises the first or second vesicle localization moiety selected from the group consisting of ACE, AD AMID, ADAMIS, ADAM9, AGRN, ALCAM, ANPEP, ANTXR2, ATP1A1, ATP1B3, BSG, BTN2A1, CALM1, CANX, CD151, CD19, CD1A, CD1B, CD1C, CD2, CD200, CD200R1, CD226, CD247, CD274, CD276, CD33, CD34, CD36, CD37, CD3E, CD40, CD40LG, CD44, CD47, CD53, CD58, CD63, CD81, CD82, CD84, CD86, CD9, CHMP1A, CHMP1B,

Cl IMP2A, CHMP3, CHMP4A, CHMP4B, CHMP5, CHMP6, CLSTN1, COL6A1, CR1, CSF1R, CXCR4, DDOST, DLL1, DLL4, DSG1, EMB, ENG, EVI2B, Fl 1R, FASN, FCER1G, FCGR2C, FLOT1, FLOT2, FLT3, FN1, GAPDH, GLG1, GRIA2, GRIA3, GYPA, HSPG2, ICAM1, ICAM2, ICAM3, IGSF8, IL1RAP, IL3RA, IL5RA, IST1, ITGA2, ITGA2B, ITGA3, ITGA4, ITGA5, ITGA6, 1TGAL, ITGAM, ITGAV, ITGAX, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, JAG1, JAG2, KIT, LAMP2, LGALS3BP, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LMAN2, LRRC25, LY75, M6PR, MFGE8, MMP14, MPL, MRC1, MVB12B, NECTIN1, N0M01, NOTCH 1, NOTCH2, NOTCFI3, NOTCH4, NPTN, NRP1, PDCD1, PDCD1LG2, PDCD6IP, PDGFRB, PECAM1, PLXNB2, PLXND1, PROMI, PTGES2, PTGFRN, PTPRA, PTPRC, PTPRJ, PTPRO, RPN1, SDC1, SDC2, SDC3, SDC4, SDCBP, SDCBP2, SELPLG, SIGLEC7, SIGLEC9, SIRPA, SLIT2, SNF8, SPN, STX3, TACSTD2, TFRC, TLR2, TMED10, TNFRSF8, TRAC, TSG101, TSPAN14, TSPAN7, TSPAN8, TYROBP, VPS25, VPS28, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS4A, VPS4B, VTI1A and VTI1B, or a variant or homologue thereof, and wherein the selected first and second vesicle localization moieties are different proteins and are not isoforms, or orthologs. In one example, the variant may be an allelic variant or an isoform. In another example, the homologue may be an ortholog or paralog. In a further specific embodiment, the surface-and-transmembrane domain of the first vesicle localization moiety of the IL6/IL6 receptor alpha complex-binding EV protein is a surface-and-transmembrane domain of LAMP2 or a variant or homologue thereof. For example, the LAMP2 may be LAMP2b or a variant or homologue thereof For example, the surface-and-transmembrane domain of LAMP2b of the IL6/IL6 receptor alpha complex-binding EV protein may comprise an amino acid sequence starting from leucine at position 29 to alanine at position 395 of NCBI Reference Sequence Accession number NP 054701.1, or an amino acid sequence of:

11 VI VI A (SEQ ID NO: XX7 (also referred to herein as SEQ ID NO: 46).

[90] In a further embodiment of the invention, the cytosolic domain of the second vesicle localization moiety of the IL6/IL6 receptor alpha complex-binding EV protein is the cytosolic domain selected from the group consisting of PTGFRN, ITGA3, IL3RA, SELPLG, ITGB1 , CLSTN1, and a variant or homologue thereof. In a specific embodiment, the cytosolic domain of the second vesicle localization moiety of the IL6/IL6 receptor alpha complex-binding EV protein may be PTGFRN or a variant or homologue thereof.

[91] Merely by way of example, in a specific embodiment of the invention, the chimeric vesicle localization moiety of the IL6/IL6 receptor alpha complex-binding EV protein comprises the surface-and-transmembrane domain of LAMP2b and the cytosolic domain of PTGFRN. For example, in one embodiment, the cytosolic domain of PTGFRN of the IL6/IL6 receptor alpha complex-binding EV protein comprises an amino acid sequence starting from serine at position 854 to aspartic acid at position 879 of NCBI Reference Sequence Accession number NP_065173.2, or an amino acid sequence of: SSHWCCKKEVQETRRERRRLMSMEMD (SEQ ID NO: XX8 (also referred to herein as SEQ ID NO: 47). In another embodiment, the chimeric vesicle localization moiety of the IL6/IL6 receptor alpha complex-binding EV protein may comprise the surface-and-transmembrane domain of LAMP2b and the cytosolic domain of PTGFRN comprising an amino acid sequence of

CKKEVQETRRERRRLMSMEMD (SEQ ID NO: XX8B (also referred to herein as SEQ ID NO: 48).

[92] In other embodiments, the chimeric vesicle localization moiety of the IL6/IL6 receptor alpha complex-binding EV protein may comprise any of the chimeric vesicle localization moieties described in WO2021/154888 (PCT/2021/015334, Mantra Bio Inc.) incorporated herein by reference.

[93] Immunoglobulin CH3 domain

[94] In another embodiment of the invention, the IL6/IL6 receptor alpha complex-binding EV protein additionally comprises a third constant immunoglobulin domain, CH3 domain, of IgGl heavy chain. For example, the IgGl heavy chain may be a human IgGl heavy chain. Merely by way example, the CH3 domain of human IgGl heavy chain may comprise an amino acid sequence starting from glycine at position 110 to lysine at position 216 of GenBank Accession Number: AAL96263. 1, or an amino acid sequence of: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: XX9 (also referred to herein as SEQ ID NO: 49).

[95] Linkers

[96] The invention further provides that the IL6/IL6 receptor alpha complex-binding EV protein may additionally comprise a flexible linker. In one embodiment, the flexible linker is a glycine-serine rich linker. In another embodiment, the glycine-serine rich linker comprises greater than 80% glycine and serine residues. In yet a further embodiment, the glycine-serine rich linker may comprise 100% glycine and serine residues. Merely by way of example, the glycine-serine rich linker may comprise a linker length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. Examples of glycine-serine rich linker include, but are not limited to, (GS)a (SEQ ID NO: XXI 0 (also referred to herein as SEQ ID NO: 50), (GS)4 (SEQ ID NO: XXI 1 (also referred to herein as SEQ ID NO: 51), (GS)s (SEQ ID NO: XX12 (also referred to herein as SEQ ID NO: 52), (GS)6 (SEQ ID NO: XX13 (also referred to herein as SEQ ID NO: 53), (GS)7 (SEQ ID NO: XX13B (also referred to herein as SEQ ID NO: 54), (GS)s (SEQ ID NO: XX14 (also referred to herein as SEQ ID NO: 55), (GS)g (SEQ ID NO: XXI 5 (also referred to herein as SEQ ID NO: 56), (GS)io (SEQ ID NO: XXI 6 (also referred to herein as SEQ ID NO: 57), (GGS)2 (SEQ ID NO: XXI 7 (also referred to herein as SEQ ID NO: 58), (GGS)₃ (SEQ ID NO: XXI 8 (also referred to herein as SEQ ID NO: 59), (GGS)4 (SEQ ID NO: XXI 9 (also referred to herein as SEQ ID NO: 60), (GGS)s (SEQ ID NO: XX20 (also referred to herein as SEQ ID NO: 61), (GGS)6 (SEQ ID NO: XX21 (also referred to herein as SEQ ID NO: 62), (GGGS)2 (SEQ ID NO: XX22 (also referred to herein as SEQ ID NO: 63), (GGGS)₃ (SEQ ID NO: XX23 (also referred to herein as SEQ ID NO: 64), (GGGS)4 (SEQ ID NO: XX24 (also referred to herein as SEQ ID NO: 65), (GGGS)s (SEQ ID NO: XX25 (also referred to herein as SEQ ID NO: 66), (GGGGS)? (SEQ ID NO: XX26 (also referred to herein as SEQ ID NO: 67), (GGGGS)₃ (SEQ ID NO: XX27 (also referred to herein as SEQ ID NO: 68), (GGGGS)₄ (SEQ ID NO: XX28 (also referred to herein as SEQ ID NO: 69), (GGGGGS) (SEQ ID NO: XX29 (also referred to herein as SEQ ID NO: 70), (GGGGGS)₂ (SEQ ID NO: XX30 (also referred to herein as SEQ ID NO: 71), (GGGGGS)₃ (SEQ ID NO: XX31 (also referred to herein as SEQ ID NO: 72), (SG)₂S (SEQ ID NO: XX32 (also referred to herein as SEQ ID NO: 73), (SG)₃S (SEQ ID NO: XX33 (also referred to herein as SEQ ID NO: 74), (SG)₄S (SEQ ID NO: XX34 (also referred to herein as SEQ ID NO: 75), (SG)₅S (SEQ ID NO: XX35 (also referred to herein as SEQ ID NO: 76), (SG)₆S (SEQ ID NO: XX36 (also referred to herein as SEQ ID NO: 77), (SG)₇S (SEQ ID NO: XX37 (also referred to herein as SEQ ID NO: 78), (SG)₈S (SEQ ID NO: XX38 (also referred to herein as SEQ ID NO: 79), and (SG)QS (SEQ ID NO: XX39 (also referred to herein as SEQ ID NO: 80), or a portion thereof. In a specific embodiment, the glycine-serine rich linker is any of (GS)₃ (SEQ ID NO: XXI 0 (also referred to herein as SEQ ID NO: 50), (GS)s (SEQ ID NO: XX12 (also referred to herein as SEQ ID NO: 52), (SG)4S (SEQ ID NO: XX34 (also referred to herein as SEQ ID NO: 75), and (GGGGS)₃ (SEQ ID NO: XX27 (also referred to herein as SEQ ID NO: 68).

[97] Signal Peptide

[98] In accordance with the practice of the invention, the IL6/IL6 receptor alpha complex- binding EV protein may additionally comprise a signal peptide at its N-terminus for insertion and passage of the GP130 protein portion and the surface domain of the chimeric vesicle localization moiety through a lipid bilayer of an extracellular vesicle or an exosomc. For example, the signal peptide may be present in a nascent or newly translated IL6/IL6 receptor alpha complex-binding EV protein. Further, in an embodiment of the invention, the signal peptide may be cleaved off during or following passage of the GP130 protein portion and the surface domain of the chimeric vesicle localization moiety through the lipid bilayer of an extracellular vesicle or exosome.

[99] Merely by way of example, the signal peptide may be a mouse Ig kappa signal peptide or functional equivalence. For example, the mouse Ig kappa signal peptide may comprise an amino acid sequence from methionine at position 1 to aspartic acid at position 21 of GenBank Accession Number: AAH80787.1, or an amino acid sequence of:

METDTLLLWVLLLWVPGSTGD (SEQ ID NO: XX40 (also referred to herein as SEQ ID NO: 81).

[100] Further, the invention provides that in one embodiment, in the extracellular vesicle or exosome, the signal peptide is absent or missing from a processed or mature IL6/IL6 receptor alpha complex-binding EV protein. In another embodiment, the extracellular vesicle or exosome comprises the processed or mature IL6/IL6 receptor alpha complex-binding EV protein.

[101] In an embodiment of the invention, the processed or mature IL6/IL6 receptor alpha complex-binding EV protein is found on the surface of the extracellular vesicle or exosome. In another embodiment, the GP130 protein portion and the surface domain of the chimeric vesicle localization moiety may be found on the outer surface or external to the extracellular vesicle or exosome, the transmembrane domain of the chimeric vesicle localization moiety may be found in the lipid bilayer of the extracellular vesicle or exosome, and the cytosolic domain of the chimeric vesicle localization moiety may be found in the inner or interior portion of the extracellular vesicle or exosome. For a complete description of the chimeric vesicle localization moiety, see

WO2021/154888 (PCT/2021/015334, Mantra Bio Inc.), incorporated by reference herein.

[102] Epitope tags

[103] The extracellular vesicles or exosomes of the invention may further comprise an epitope tag e.g. located in the IL6/IL6 receptor alpha complex. Merely by way of example, the epitope tags include but are not limited to a FLAG tag, a myc tag, a human influenza hemagglutinin (HA) tag, and a (His)e tag. In one embodiment, the FLAG tag comprises an amino acid sequence of DYKDDDDK (SEQ ID NO: XX41 (also referred to herein as SEQ ID NO: 82). In another embodiment, the myc tag comprises an amino acid sequence of EQKLISEEDL (SEQ ID NO: XX42 (also referred to herein as SEQ ID NO: 83). In a further embodiment, the HA tag comprises an amino acid sequence of YPYDVPDYA (SEQ ID NO: XX43 (also referred to herein as SEQ ID NO: 84), In yet a further embodiment, the (His)e tag comprises an amino acid sequence of IIHHHHH (SEQ ID NO: XX44 (also referred to herein as SEQ ID NO: 85).

[104] Also, the invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the nascent or newly translated IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-signal peptide-human GP130 portion-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex and wherein an epitope tag may be present optionally at cither N-terminus between the signal peptide and human GP130 portion or at C -terminus after the cytosolic domain of human PTGFRN.

[105] The invention additionally provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-human GP130 portion-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus (mature or processed IL6/IL6 receptor alpha complex-binding EV protein), following incorporation into an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex and wherein an epitope tag may be present optionally at either N-terminus before the human GP130 portion or at C-terminus after the cytosolic domain of human PTGFRN.

[106] The invention further provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the nascent or newly translated IL6/1L6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-signal peptide-human GP130 portion-linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex and wherein an epitope tag may be present optionally at either N-terminus between the signal peptide and human GP130 portion or at C-terminus after the cytosolic domain of human PTGFRN.

[107] The invention additionally provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-human GP130 portion-linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, (mature or processed IL6/IL6 receptor alpha complex-binding EV protein), following incorporation into an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex and wherein an epitope tag may be present optionally at either N-terminus before the human GP130 portion or at C -terminus after the cytosolic domain of human PTGFRN.

[108] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the nascent or newly translated IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-signal peptide-human GP130 portion-linker-CH3 domain of human IgGl -linker-human Lamp2b extracellular domain-and- transmembrane domain-cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex and wherein an epitope tag may be present optionally at either N-terminus between the signal peptide and human GP130 portion or at C -terminus after the cytosolic domain of human PTGFRN.

[109] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-human GP130 portion-linker-CI I3 domain of human IgGl- linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, (mature or processed IL6/IL6 receptor alpha complex -binding EV protein), following incorporation into an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex and wherein an epitope tag may be present optionally at either N-terminus before the human GP130 portion or at C -terminus after the cytosolic domain of human PTGFRN.

110] In some embodiments, the nascent or newly translated VEGF-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-signal peptide-VEGFR-domain fusion units-1 inker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome, wherein an epitope tag may be present optionally at either N-terminus between the signal peptide and the first VEGFR-domain fusion unit or at C -terminus after the cytosolic domain of human PTGFRN. In some embodiments, the VEGF-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-VEGFR-domain fusion units-linker-human Lamp2b extracellular domain-and- transmembrane domain-cytosolic domain of human PTGFRN-C terminus, (mature or processed VEGF-binding EV protein), following incorporation into an extracellular vesicle or exosome, wherein an epitope tag may be present optionally at either N -terminus before the first VEGFR- domain fusion unit or at C -terminus after the cytosolic domain of human PTGFRN.

[111] GP130 configurations

112] In some embodiments, the extracellular vesicles or exosomes of the invention contains GP130 domain one to three (1-3) protein portion with or without additional GP130 domain four (4), four to five (4-5), or four to six (4-6) linked directly through its C-terminus to the N- terminus of the chimeric vesicle localization moiety. In some embodiments, in the extracellular vesicle or exosome of the invention, the IL6/IL6 receptor alpha complex-binding EV protein is free of GP130 transmembrane domain. In further embodiments, in the extracellular vesicle or exosome of the invention, the IL6/IL6 receptor alpha complex-binding EV protein is free of GP130 cytoplasmic domain. In yet further embodiments, in the extracellular vesicle or exosome of the invention, the IL6/IL6 receptor alpha complex-binding EV protein is free of both GP130 transmembrane domain and cytoplasmic domain.

[113] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the nascent or newly translated IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-signal peptide-human GP130 portion-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex.

[114] The invention further provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the IL6/IL6 receptor alpha complex-binding EV protein from amino-to- carboxyl direction is organized as: N terminus-human GP130 portion-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus (mature or processed IL6/IL6 receptor alpha complex-binding EV protein), following incorporation into an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex. 115] In some embodiments, the IL6/IL6 receptor alpha complex-binding EV protein comprises an amino acid sequence of processed or mature MB20 from amino acid at position 22 to end of SEQ ID NO: 20 or processed or mature MB22 from amino acid at position 22 to end of SEQ ID NO: 22.

[116] In some embodiments, the GP130 domain 1-3 protein portion with or without additional GP130 domain 4, 4-5, or 4-6 is linked through a glycine-serine rich linker to the N- terminus of the chimeric vesicle localization moiety. Merely by way of example, the glycine-serine rich linker is (GGGGS)3 (SEQ ID NO: XX27 (also referred to herein as SEQ ID NO: 68).

[117] Further, the invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the nascent or newly translated IL6/IL6 receptor alpha complex- binding EV protein from amino-to-carboxyl direction is organized as: N terminus-signal peptide- human GP130 portion-linker-human Lamp2b extracellular domain-and-transmembrane domain- cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex.

[118] Additionally, the invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-human GP130 portion-linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, (mature or processed IL6/IL6 receptor alpha complex-binding EV protein), following incorporation into an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex.

[ 119] In some embodiments, the IL6/IL6 receptor alpha complex-binding EV protein comprises an amino acid sequence of a mature or processed MB 16 from amino acid at position 22 to end of SEQ ID NO: 16 or a mature or processed MB 18 from amino acid at position 22 to end of SEQ ID NO: 18.

[120] 72) In some embodiments, the GP130 domain 1-3 protein portion with or without additional GP130 domain 4, 4-5, or 4-6 is linked to the N-terminus of the chimeric vesicle localization moiety through glycine-rich linkers attached to both ends of the CH3 domain of human IgGl heavy chain. For example, the glycine-rich linkers attached to both ends of the CI 13 domain of human IgGl heavy chain may comprise the (GS)3 linker (SEQ ID NO: XX 10 (also referred to herein as SEQ ID NO: 50) attached to the N-terminus of the CH3 domain and the (GS)s linker (SEQ ID NO: XX12 (also referred to herein as SEQ ID NO: 52) attached to the C -terminus of the CH3 domain.

[121] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the nascent or newly translated IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-signal peptide-human GP130 portion-linker-CH3 domain of human IgGl -linker-human Lamp2b extracellular domain-and- transmembrane domain-cytosolic domain of human PTGFRN-C terminus, prior to incorporation onto the surface of an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex.

[122] In some embodiments, the IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-human GP130 portion-linker-CH3 domain of human IgGl -linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, (mature or processed IL6/IL6 receptor alpha complex- binding EV protein), following incorporation into an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex.

[123] In some embodiments, the IL6/IL6 receptor alpha complex-binding EV protein comprises an amino acid sequence of a mature or processed MB02 from amino acid at position 22 to end of SEQ ID NO: 2, a mature or processed MB04 from amino acid at position 22 to end of SEQ ID NO: 4, a mature or processed MB06 from amino acid at position 22 to end of SEQ ID NO: 6, or a mature or processed MB08 from amino acid at position 22 to end of SEQ ID NO: 8.

[124] The invention provides further embodiments of the extracellular vesicles or exosomes of the invention, wherein the IL6/IL6 receptor alpha complex-binding EV protein from amino-to- carboxyl direction is organized as: N terminus-signal peptide-human GP130 domain 1-5 portion- (GS)a linker-CH3 domain of human IgGl-(GS)s linker-human Lamp2b extracellular domain-and- transmembrane domain-cytosolic domain of human PTGFRN-C terminus, wherein the signal peptide initially present in the nascent or newly synthesized protein is cleaved off in the processed or mature protein and wherein the GP130 domain 1 portion comprises T102Y, QI 13F and N1 I4E to increase binding affinity for IL6/IL6 receptor alpha complex.

[125] Specific Embodiments Of The Extracellular Vesicles Of The Invention

[126] The invention provides embodiments of the extracellular vesicles or exosomes of the invention, wherein the VEGF-binding EV protein comprises the Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) comprises an amino acid sequence from serine at position 129 to isoleucine at position 230 ofNCBI Reference Sequence Accession Number: NP_002010.2 or UniProtKB Accession Number: P17948-1, (both incorporated by reference) or an amino acid sequence of: SDTGRPFVEMYSEI PEI IHMTEGRELVI PCRVTSPNITVTLKKFPLDTLI PDGKRI IWDS

RKGFI ISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTII (SEQ ID NO: XX45 (also referred to herein as SEQ ID NO: 86), or an amino acid sequence having 90% sequence identity thereof. [127] The invention provides extracellular vesicles or exosomes of the invention, , comprising: (a) the IL6/IL6 receptor alpha complex-binding EV protein is selected from the group consisting of a mature or processed MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, a mature or processed MB06 comprising an amino acid sequence from position 22 to end of SEQ ID NO:6, a mature or processed MB08 comprising an amino acid sequence from position 22 to end of SEQ ID NO:8, a mature or processed MB 16 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 16, a mature or processed MB 18 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 18, a mature or processed MB20 comprising an amino acid sequence from position 22 to end of SEQ ID NO:20, and a mature or processed MB22 comprising an amino acid sequence from position 22 to end of SEQ ID NO:22, and (b) the VEGF-binding EV protein is selected from the group consisting of a mature or processed MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10, a mature or processed MB 12 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 12, a mature or processed MB 14 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 14, and a mature or processed MB25 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 26.

[128] In a specific embodiment, extracellular vesicle or exosome of the invention comprises: (a) the IL6/IL6 receptor alpha complex-binding EV protein selected from the group consisting of a mature or processed MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, a mature or processed MB08 comprising an amino acid sequence from position 22 to end of SEQ ID NO:8, a mature or processed MB16 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 16, and a mature or processed MB20 comprising an amino acid sequence from position 22 to end of SEQ ID NO:20, and (b) the VEGF-binding EV protein selected from the group consisting of a mature or processed MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10, a mature or processed MB 12 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 12, a mature or processed MB 14 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 14, and a mature or processed MB25 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 26.

[129] In another specific embodiment of the invention, the extracellular vesicle or exosome comprises: (a) the IL6/IL6 receptor alpha complex-binding EV protein, a mature or processed MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, and (b) the VEGF- binding EV protein selected from the group consisting of a mature or processed MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10, a mature or processed MB 12 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 12, a mature or processed MB 14 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 14, and a mature or processed MB25 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 26.

[130] In a further specific embodiment, the extracellular vesicle or exosome comprises: (a) the IL6/IL6 receptor alpha complex-binding EV protein selected from the group consisting of a mature or processed MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, a mature or processed MB08 comprising an amino acid sequence from position 22 to end of SEQ ID NO:8, a mature or processed MB 16 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 16, and a mature or processed MB20 comprising an amino acid sequence from position 22 to end of SEQ ID NO:20, and (b) the VEGF-binding EV protein, a mature or processed MB25 (SEQ ID NO: 39).

[131] In a further embodiment, the extracellular vesicle or exosome of comprises: (a) the IL6/IL6 receptor alpha complex-binding EV protein selected from the group consisting of a mature or processed MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, a mature or processed MB08 comprising an amino acid sequence from position 22 to end of SEQ ID NO:8, a mature or processed MB 16 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 16, and a mature or processed MB20 comprising an amino acid sequence from position 22 to end of SEQ ID NO:20, and (b) the VEGF-binding EV protein, a mature or processed MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10.

[132] In a further specific embodiment, the extracellular vesicle or exosome comprises: (a) the IL6/IL6 receptor alpha complex-binding EV protein, a mature or processed MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, and (b) the VEGF-binding EV protein, a mature or processed MB25 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 26. [133] Additionally, in another specific embodiment, the extracellular vesicle or exosome comprises: (a) the IL6/IL6 receptor alpha complex-binding EV protein, a mature or processed MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, and (b) the VEGF- binding EV protein, a mature or processed MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10. For example, in some embodiments, the invention provides compositions comprising a plurality of extracellular vesicle or exosome having about 75% or more of the extracellular vesicles and/or exosomes therein that are positive for both the mature or processed MB02 and the mature or processed MB010 proteins.

[134] In some embodiments, mode of IL6/IL6 receptor alpha complex-binding EV protein molecules per extracellular vesicle and/or exosome positive for the IL6/IL6 receptor alpha complex- binding EV protein is at about 120 molecules.

[135] In some embodiments, vesicle flow cytometry is used to determine abundance of the VEGF-binding EV protein within an extracellular vesicle and/or exosome population.

[136] In some embodiments, the IL6/IL6 receptor alpha complex-binding EV protein binds soluble 1L6-IL6 receptor complex inhibiting pro-inflammatory IL6-trans-signaling.

[137] Compositions comprising EVs or exosomes of the invention

[138] The invention provides pharmaceutical compositions for inhibiting IL6 trans- signaling and for inhibiting VEGF-signaling comprising a plurality of extracellular vesicle or exosome of the invention and pharmaceutically acceptable excipients.

[139] In one embodiment, the composition comprises extracellular vesicles or exosomes inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 8.22E+08 EV/mL to 3.22E+10 EV/mL. In another embodiment, the extracellular vesicles or exosomes inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 1.73E+10 EV/mL. In a further embodiment, the extracellular vesicles or exosomes inhibit IL6-trans-signaling in FIUVEC cells with an IC50 of about 8.22E+08 EV/mL.

[140] The invention further provides compositions comprising a plurality of extracellular vesicles or exosomes of the invention, wherein the VEGF-binding EV protein binds VEGF inhibiting VEGF-signaling or angiogenesis. In one embodiment, the extracellular vesicles or exosomes inhibit VEGF signaling in HUVEC cells with an IC50 of about 1.95E+09 EV/mL to 6.88E+09 EV/mL. In another embodiment, the extracellular vesicles or exosomes inhibit VEGF signaling in HUVEC cells with an IC50 of about 1.95E+09 EV/mL to 2.81 E+09 E V/mL. In an additional embodiment, the composition comprises extracellular vesicles or exosomes which inhibit VEGF signaling in HUVEC cells with an IC50 of about 2.81E+09 EV/mL. Also, in a yet further embodiment, the extracellular vesicles or exosomes inhibit VEGF signaling in HUVEC cells with an IC50 of about 1.95E+09 EV/mL.

[141] The invention additionally provides composition comprising a plurality of extracellular vesicle or exosome which simultaneously binds both IL6-IL6 receptor complex inhibiting IL6-trans-signaling and binds VEGF inhibiting VEGF-signaling. In an embodiment of the invention, the extracellular vesicles or exosomes bind multiple IL6-IL6 receptor complexes and multiple copies of VEGF. Merely as an example, the extracellular vesicles or exosomes of the composition inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 3.22E+10 EV/mL and VEGF signaling in HUVEC cells with an IC50 of about 2.81E+09 EV/mL. In another embodiment, the extracellular vesicles or exosomes inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 3.22E+10 EV/mL and VEGF signaling in HUVEC cells with an IC50 of about 1.95E+09 EV/mL. Further, in yet another embodiment, the extracellular vesicles or exosomes inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 8.22E+08 EV/mL and VEGF signaling in HUVEC cells with an IC50 of about 2.81E+09 EV/mL. Further still, the invention provides an embodiment of the composition, wherein the extracellular vesicles or exosomes inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 8.22E+08 EV/mL and VEGF signaling in HUVEC cells with an IC50 of about 1 .95E+09 EV/mL.

[ 142] In the composition, in some embodiments, the extracellular vesicle or exosome contains VEGF -binding EV proteins which comprises three (3) VEGFR-domain fusion units, so as to maintain VEGF function in the presence of the IL6/IL6 receptor alpha complex-binding EV protein.

[143] In accordance with the invention, an extracellular vesicle or exosome of the invention can be a membrane that encloses an internal space. Extracellular vesicles can be cell-derived bubbles or vesicles made of the same material as cell membranes, such as phospholipids. Cell-derived extracellular vesicles can be smaller than the cell from which they are derived and range in diameter from about 20 nm to 1000 nm (c.g., 20 nm to 1000 nm; 20 nm to 200 nm; 90 nm to 150 nm). Such vesicles can be created through the outward budding and fission from plasma membranes, assembled at and released from an endomembrane compartment, or derived from cells or vesiculated organelles having undergone apoptosis, and can contain organelles. They can be produced in an endosome by inward budding into the endosomal lumen resulting in intraluminal vesicles of a multivesicular body (MVB) and released extracellularly as exosomes upon fusion of the multivesicular body (MVB) with the plasma membrane. They can be derived from cells by direct and indirect manipulation that may involve the destruction of said cells. They can also be derived from a living or dead organism, an explanted tissue or organ, and/or a cultured cell.

[144] Examples of extracellular vesicles include exosomes, ectosome, microvesicle, microsome or other cell-derived membrane vesicles. Other cell-derived membrane vesicles include a shedding vesicle, a plasma membrane-derived vesicle, and/or an exovesicle.

[145] An “extracellular vesicle” used here is produced by cells, and may comprise a phospholipid membrane bilayer enclosing a luminal space. The membrane bilayer incorporates proteins and other macromolecules derived from the cell of origin. The luminal space encapsulates lipids, proteins, organic molecules and macromolecules including nucleic acids and polypeptides.

[146] Exosomes can be secreted membrane-enclosed vesicles that originate from the endosome compartment in cells. The endosome compartment, or the multi-vesicular body, can fuse with the plasma membrane of the cell, with ensuing release to the extracellular space of their vesicles as exosomes. Further, an exosome can comprise a bilayer membrane, and can comprise various macromolecular cargo either within the internal space, displayed on the externa! surface of the extracellular vesicle, and/or spanning the membrane. Cargo can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and/or combinations thereof. Exosomes can range in size from about 20 nm to about 300 nm. Additionally, the exosome may have an average diameter in the range of about 50 nm to about 220 nm. Preferably, in a specific embodiment, the exosome has an average diameter of about 120 nm + 20 nm.

[147] In some instances, exosomes and other extracellular vesicles can be characterized and marked based on their protein compositions, such as integrins and tetraspanins. Other protein markers that are used to characterize exosomes and other extracellular vesicles (EVs) include TSG101, ALG-2 interacting protein X (ALIX), flotillin 1, and cell adhesion molecules which are derived from the parent cells in which the exosome and/or EV is formed. Similar to proteins, lipids can be major components of exosomes and EVs and can be utilized to characterize them.

[148] An extracellular vesicle can have a longest dimension, such as a cross-sectional diameter, of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 nm and/or at most about 1000, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 nm. In some instances, a longest dimension of a vesicle can range from about 10 nm to about 1000 nm, about 20 nm to about 1000 nm, about 30 nm to about 1000 nm, about 10 nm to about 100 nm, about 20 nm to about 100 nm, about 30 nm to about lOOnm, about 40 nm to about 100 nm, about 10 nm to about 200 nm, about 20 nm to about 200 nm, about 30 nm to about 200 nm, about 40 nm to about 200 nm, about 10 nm to about 120 nm, about 20 nm to about 120 nm, such as about 30 nm to about 120 nm, about 40 nm to about 120 nm, about 10 nm to about 300 nm, about 20 nm to about 300 nm, about 30 nm to about 300 nm, about 40 nm to about 300 nm, about 50 nm to about 1000 nm, about 500 nm to about 2000 nm, about 100 nm to about 500 nm, about 500 nm to about 1000 nm, and such as about 40 nm to about 500 nm, each range inclusive. When referring to a plurality of vesicles, such ranges can represent the average of all vesicles, including naturally occurring and modified vesicles in the mix. [149] As used herein, the term “average” may be mean, mode or medium for a group of measurements.

[150] As used herein, the term “about” when used before a numerical designation, e.g. , diameter, size, temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by (+) or (-) 10 %, 5 % or 1 %.

[151] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the culture" includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth.

[152] Without being bound by any theory, a “vesicle localization moiety” (also referred to as a vesicle targeting moiety) may be a macromolecule that localizes at an extracellular vesicle. -In a preferred embodiment, the vesicle localization moiety is a transmembrane protein comprising a surface domain, a transmembrane domain and a cytosolic domain. Localization of such a transmembrane protein at an extracellular vesicle results in the surface domain at the outer surface of the vesicle, the transmembrane domain with the lipid bilayer of the vesicle and the cytosolic domain in the lumen of the vesicle. Because of topological equivalence, a surface domain may also be referred to as an extracellular domain, since the surface domain on the surface of an exosome shares the same topological state as plasma membrane bound transmembrane protein on the surface of a cell; similarly, a cytosolic domain may be referred to as a lumenal domain, since part of the cytoplasm where the cytosolic domain initially resides is incorporated into the lumen of a vesicle produced by inward budding of an endosomal membrane to eventually produce multiple intraluminal vesicles of a multivesicular body (MVB) prior to secretion of the vesicles as exosomes upon fusion of the MVB with the plasma membrane of an EV producer cell.

[153] The vesicle localization moiety may have a surface domain, a transmembrane domain and a cytosolic domain. A complete description may be found in WO2021/154888 (PCT/2021/015334, Mantra Bio Inc., incorporated by reference herein). Such protein domains are known in the art and are well annotated and defined for the proteins described, herein, in the figures and in annotations associated with Accession Numbers from publicly available databases, referred herein, such as UniProtKB (UniProt Release 2019_11 (11 -Dec-2019); The UniProt Consortium (2019) UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2019 Jan 08;47(Dl):D506-515), Reference sequence (RefSeq) database at NCBI (Nucleic Acids Res. 2016 Jan 4;44(Dl):D733-45), GenBank database (Nucleic Acids Res. 2020 Jan 08;48(Dl):D84-86), and Genome Reference Consortium Human Build 38 patch release 13 (GRCh38.pl 3; GenBank assembly accession GCA_000001405.28 and RefSeq assembly accession GCF_000001405.39).

[154] A “chimeric vesicle localization moiety” is a vesicle localization moiety which may be produced by substituting one vesicle localization domain with another vesicle localization domain, so as to produce a chimeric vesicle localization moiety. A complete description may be found in WO2021/154888 (PCT/2021/015334, Mantra Bio Inc., incorporated by reference herein). A chimeric vesicle localization moiety may be obtained by combining one or more functional domains of one vesicle localization moiety with one or more functional domains of another, different vesicle localization moiety. The combination comprises portion(s) of at least two vesicle localization moieties, so as to obtain a chimeric vesicle localization moiety which is superior in its association with an EV than either of the parental vesicle localization moiety, as quantified by mean recombinant protein density on EV surface and/or fraction (or percent) of total EVs positive for the recombinant protein. In an embodiment, the chimeric vesicle localization moiety comprises a surface domain, a transmembrane domain and a lumenal or cytosolic domain of a transmembrane protein or the two parental transmembrane proteins from which it is derived. In an embodiment, the chimeric vesicle localization moiety has the same arrangement of surface domain, transmembrane domain and lumenal or cytosolic domain as described for the vesicle localization moiety, described above. Merely by way of example, a chimeric vesicle localization moiety comprising a surface-and- transmembrane domain of a first vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety may interact synergistically to increase accumulation at an extracellular vesicle. This not only may improve EV localization but may also change the composition of EVs.

[155] Surface domain” is a subset of the protein or polypeptide primary sequence that is exposed to the extra-EV environment. The surface domain may be a loop between two transmembrane domains or it can contain one of the termini (amino or carboxy) of the protein. Protein domain topology relative to the membrane bi-layer can be determined empirically by assessing what portions of the protein are digested by an external protease. More recently, characteristic amino acid patterns, such as basic or acidic residues in the juxta-membrane regions of the protein have been used to algorithmically assign probable topologies (extracellular versus cytosolic) to integral membrane proteins. Since EVs have the same membrane topology orientation as the plasma membrane of the whole cell (the outer leaflet of the membrane is the same between cells and EVs), these algorithms can be applied to EV resident proteins as well. As such, the surface domain of an EV localizing transmembrane protein may sometimes be referred to as an extracellular domain due to the same membrane topology of an EV and plasma membrane. For example, the “surface domain” may be a short peptide of approximately 10-15 amino acids. In one embodiment, the “surface domain” may be an unstructured polypeptide. In another embodiment, the “surface domain” is the entire surface domain of an integral membrane protein. In yet another embodiment, the “surface domain” is part or portion of the surface domain of an integral membrane protein. In an embodiment, the surface domain is amino terminal to the transmembrane domain and cytosolic domain. In an embodiment, the surface domain is at the N -terminus of the vesicle localization moiety or the chimeric vesicle localization moiety and is on the external surface of an extracellular vesicle, such as an exosome.

[156] “Transmembrane domain” may be a span of about 18-40 aliphatic, a polar and hydrophobic amino acids that assembles into an alpha-helical secondary structure and spans from one face of a membrane bilayer to the other face, meaning that the N-terminus of the helix extends at least to and in many cases beyond the phospholipid headgroups of one membrane leaflet while the C-terminus extends to the phospholipid headgroups of the other leaflet. In an embodiment, the transmembrane domain connects an amino terminal surface domain with a carboxyl terminal cytosolic domain.

[157] “Cytosolic domain” is a subset of the protein or polypeptide primary sequence that is exposed to the intra-EV or intracellular environment. The cytosolic domain can be a loop between two transmembrane domains or it can contain one of the termini (amino or carboxy) of the protein. Its topology is distinct from that of the transmembrane and the surface domains. In an embodiment, the cytosolic domain is in the cytoplasmic side of a cell. In another embodiment, the cytosolic domain is in the lumen of a vesicle. In an embodiment, the cytosolic domain is at the C-terminus of the vesicle localization moiety or the chimeric vesicle localization moiety

[158] Merely by way of example, sequences corresponding to “surface domain,” “transmembrane domain” and “cytosolic domain” for the proteins disclosed herein may be found within the description under protein accession numbers provided herein. Particularly useful examples are the proteins cataloged within UniProtKB (UniProt Release 2019 11 (11 -Dec-2019)) (incorporated by reference) where under each accession number amino acid sequence along with features and functional domains are provided

[159] In a preferred embodiment, a “chimeric vesicle localization moiety” comprises the

“surface-and-transmembrane domain” of one vesicle localization moiety and the “cytosolic domain” of a second vesicle localization moiety, wherein the two vesicle localization moieties are different and distinct proteins and are not isoforms. In an embodiment, the “chimeric vesicle localization moiety” comprises the “surface-and-transmembrane domain” of one vesicle localization moiety and the “cytosolic domain” of a second vesicle localization moiety, wherein the two vesicle localization moieties are different and distinct proteins and are not isoforms. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two distinct genes which are not allelic or homologs. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two distinct genes which are not orthologs. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two distinct genes which are not paralogs. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two distinct genes which are paralogs. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two nonhomologous genes. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two or more proteins encoded by two or more nonhomologous genes. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two or more proteins encoded by two or more nonhomologous human genes. In an embodiment, the “chimeric vesicle localization moiety” is produced from combining domains of two or more human genes encoding transmembrane proteins. In a preferred embodiment, the “chimeric vesicle localization moiety” is produced from combining two nonhomologous human genes or two human genes not placed within the same gene family, wherein the genes encode transmembrane proteins.

[160] “Surface-and-transmembrane domain” is a contiguous polypeptide containing both a domain that is exposed to extracellular or extra-EV solvent and a transmembrane domain as described above. [161] As used herein “isolated” means a state following one or more purifying steps but does not require absolute purity. “Isolated” extracellular vesicle (e.g., exosome) or composition thereof means an extracellular vesicle, exosome or composition thereof passed through one or more purifying steps that separate the vesicle, extracellular vesicle, exosome or composition from other molecules, materials or cellular components found in a mixture or outside of the vesicle, extracellular vesicle or exosome or found as part of the composition prior to purification or separation. Isolation and purification may be achieved in accordance with conventional methods of recombinant synthesis or cell free protein synthesis. Separation procedures of interest include affinity chromatography. Affinity chromatography makes use of the highly specific binding sites usually present in biological macromolecules, separating molecules on their ability to bind a particular ligand. For example, covalent bonds attach the ligand to an insoluble, porous support medium in a manner that overtly presents the ligand to the protein sample, thereby using natural biospecific binding of one molecular species to separate and purify a second species from a mixture. Antibodies may be used in affinity chromatography. Preferably a microsphcrc or matrix is used as the support for affinity chromatography. Such supports are known in the art and are commercially available and include activated supports that can be combined to the linker molecules. For example, Affi-Gel supports, based on agarose or polyacrylamide are low pressure gels suitable for most laboratory-scale purifications with a peristaltic pump or gravity flow elution. Affi-Prep supports, based on a pressure-stable macroporous polymer, may be suitable for preparative and process scale applications. Isolation may also be performed using methods involving centrifugation, filtration, size exclusion chromatography and vesicle flow cytometry.

[58] In general, “sequence identity” or “sequence homology,” refer to a nucleotide-to- nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. As used herein, "sequence identity" or "identity" refers, in the context of two nucleic acid sequences or amino acid sequences, to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[59] As used herein, "percent sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein (the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence which does not comprise additions or deletions comprises) can for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleotide or amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result by 100 to determine the percentage of sequence identity.

[60] Sequence comparisons, such as for the purpose of assessing identities, may be performed by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionally with default settings; Needleman, S. B. and Wunsch, C. D. (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48:443-53), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings; Altschul, S. F. et al. (1990) Basic local alignment search tool. J. Mol. Biol. 215:403-410; and Altschul, S. F. et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402), and the Smith-Waterman algorithm (see, e.g., the EMBOSS Waler aligner available at www.cbi.ac.uk/Tools/psa/cmboss_watcr/, optionally with default settings; Smith, T.F. and Waterman, M.S. (1981 ) Identification of common molecular subsequences. J. Mol. Biol. 147: 195-7). Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.

[61] The “percent identity” between two sequences may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program can be based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program can define identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the sequences being compared. Default parameters can be provided to optimize searches with short query sequences, for example, with the BLASTP program. The program can also allow use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton, J. C. and Federhen, S. (1993) Computers Chem. 17: 149-163. High sequence identity can include sequence identity in ranges of sequence identity of approximately 80% to 99% and integer values there between.

[62] A “homolog” or “homologue” can refer to any sequence that has at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence homology to another sequence. Preferably, a homolog or homologue refers to any sequence that has at least about 98%, 99%, or 99.5% sequence homology to another sequence. In some cases, the homolog can have a functional or structural equivalence with the native or naturally occurring sequence. In some cases, the homolog can have a functional or structural equivalence with a domain, a motif or a part of the protein, that is encoded by the native sequence or naturally occurring sequence.

[63] Homology comparisons may be conducted with sequence comparison programs. Computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. Sequence homologies may be generated by any of a number of computer programs, for example BLAST or PASTA, etc. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux, J. et al. (1984) Nucleic Acids Res. 12:387). Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel, F. M. et al. (1999) Short Protocols in Molecular Biology, 4^th Ed. - Chapter 18), PASTA (Atschul, S. F. et al. (1990) J. Mol. Biol. 215:403- 410) and the GENEWORKS suite of comparison tools.

[64] Percent homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments can be performed over a relatively short number of residues.

[65] In an otherwise identical pair of sequences, one insertion or deletion may cause the following amino acid or nucleotide residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, the sequence comparison method can be designed to produce optimal alignments that take into consideration possible insertions and deletions without unduly penalizing the overall homology or identity score. This can be achieved by inserting "gaps" in the sequence alignment to try to maximize local homology or identity. [66] BLAST 2 Sequences is another tool that can be used for comparing protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and the website of the National Center for Biotechnology information at the website of the National Institutes for Health).

[67] Homologous sequences can also have deletions, insertions or substitutions of amino acid residues which result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids may be grouped together based on the properties of their side chains alone.

[68] Substantially homologous sequences of the present invention include variants of the disclosed sequences, e.g., those resulting from site-directed mutagenesis, as well as synthetically generated sequences. In some cases, the variants may be allelic variants due to different alleles. In some cases, the variants may be derived from the same gene or allele due to alternative transcription start site or alternative splicing, resulting in variants which are isoforms.

Fusion Proteins

[69] The invention additionally provides IL6/1L6 receptor alpha complex-binding extracellular vesicle (EV) proteins comprising: (a) a GP130 domain 1-3 protein portion for binding to IL6/IL6 receptor alpha complex, and (b) a chimeric vesicle localization moiety comprising: (i) a surface-and -transmembrane domain of a first vesicle localization moiety and (ii) a cytosolic domain of a second vesicle localization moiety. In a further embodiment, the IL6/IL6 receptor alpha complex-binding EV protein additionally comprises GP130 domain 4, 4-5, or 4-6 such that the GP130 domain 1-3 protein portion is extended in the carboxyl direction resulting in a GP130 domain 1-4, 1-5, or 1-6 protein portion, respectively, of a full length GP130 protein.

[70] Also, the invention provides a VEGF-binding EV protein comprising: (a) at least two VEGFR-domain fusion units, and (b) a chimeric vesicle localization moiety comprising: (i) a surface-and-transmembrane domain of a first vesicle localization moiety and (ii) a cytosolic domain of a second vesicle localization moiety, wherein each VEGFR-domain fusion unit comprising Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) linked to Ig-like domain 3 of VEGF receptor 2

( VEGFR-2) for binding VEGF. [71] Methods for making such fusion proteins and for targeting/localizing fusion proteins to exosomes may be as described, e.g., in Limoni SK, et al, Appl Biochcm Biotechnol. 2018 Jun 28, doi: 10.1007/sl2010-018-2813-4.

Nudeic Acids

[72] The production of engineered vesicles can involve generation of nucleic acids that encode, at least, in part, one or more of the IL6/IL6 receptor alpha complex-binding extracellular vesicle (EV) proteins described herein.

[73] The disclosure includes vectors. Methods which are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriplional/translational control signals. Generally, expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the protein.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular expression system, e.g. mammalian cell, bacterial cell, cell- free synthesis, etc. The control sequences that are suitable for prokaryote systems, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cell systems may utilize promoters, polyadenylation signals, and enhancers.

[74] The nucleic acids may be natural, synthetic or a combination thereof. The nucleic acids may be RNA, mRNA, DNA or cDNA. Nucleic acid encoding the protein may be produced using known synthetic techniques, incorporated into a suitable expression vector using well established methods to form a protein-encoding expression vector which is introduced into a cell for protein expression using known techniques, such as transfection, lipofection, transduction and electroporation. The nucleic acids may be isolated and obtained in substantial purity. Usually, the nucleic acids, either as DNA or RNA, will be obtained substantially free of other naturally-occurring nucleic acid sequences, generally being at least about 50%, usually at least about 90% pure and are typically “recombinant,” e.g. , flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

[75] Expression of the nucleic acids can be regulated by their own or by other regulatory sequences known in the art. The nucleic acids of the invention can be introduced into suitable host cells using a variety of techniques available in the art. The expressed protein may localize or form an exosome or extracellular vesicle and released from the producing cell. Such exosomes or extracellular vesicles may be harvested from the culture medium. Similarly, the selected protein may be produced using recombinant techniques, or may be otherwise obtained, and then may be introduced directly into isolated exosomes by electroporation or transfection e.g. electroporation, transfection using cationic lipid-based transfection reagents, and the like.

[76] The nucleic acids can also include expression vectors, such as plasmids, or viral vectors, or linear vectors, or vectors that integrate into chromosomal DNA. Expression vectors can contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of cells. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria. In eukaryotic host cells, e.g., mammalian cells, the expression vector can be integrated into the host cell chromosome and then replicate with the host chromosome or the expression vector may be an episome and replicate autonomously independent of the host chromosome.

[77] Expression vectors also can contain a selection gene, also termed a selectable marker. The selection gene can encode a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the selective culture medium. Selection genes can encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, G418, puromycin, hygromycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. An exemplary selection scheme can utilize a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene can produce a protein conferring drug resistance and thus survive the selection regimen. Other selectable markers for use in bacterial or eukaryotic (including mammalian) systems are well-known in the art.

[78] When the nucleic acid is introduced into a cell ex vivo, the nucleic acid may be combined with a substance that promotes transference of a nucleic acid into a cell, for example, a reagent for introducing a nucleic acid such as a liposome or a cationic lipid, in addition to any additional excipients. Electroporation applying voltages in the range of about 20-1000 V/cm may be used to introduce nucleic acid or protein into exosomes. Transfection using cationic lipid-based transfection reagents such as, but not limited to, Lipofectamine® MessengerMAX™ Transfection Reagent, Lipofectamine® RNAiMAX Transfection Reagent, Lipofectamine® 3000 Transfection Reagent, or Lipofectamine® LTX Reagent with PLUS™ Reagent, may also be used. The amount of transfection reagent used may vary with the reagent, the sample and the cargo to be introduced. Alternatively, a vector carrying the nucleic acid of the present invention can also be used. Particularly, a composition in a form suitable for administration to a living body which contains the nucleic acid of the present invention carried by a suitable vector can be suitable for in vivo gene therapy.

[79] The nucleic acid constructs can include linker peptides. The linker peptides can adopt a helical, β-strand, coil-bend or turn conformations. The linker motifs can be flexible linkers, rigid linkers or cleavable linkers. The linker peptides can be used for increasing the stability or folding of the peptide, avoid steric clash, increase expression, improve biological activity, enable targeting to specific sites in vivo, or alter the pharmacokinetics of the resulting fusion peptide by increasing the binding affinity of the targeting domain for its receptor. Folding, as used herein, refers to the process of forming the three-dimensional structure of polypeptides and proteins, where interactions between amino acid residues act to stabilize the structure. Non-covalent interactions are important in determining structure, and the effect of membrane contacts with the protein may be important for the correct structure. For naturally occurring proteins and polypeptides or derivatives and variants thereof, the result of proper folding is typically the arrangement that results in optimal biological activity, and can conveniently be monitored by assays for activity, e.g. ligand binding, enzymatic activity, etc.

[80] Specifically, the invention provides nucleic acids encoding the IL6/IL6 receptor alpha complex-binding EV protein of the invention including a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and 7. In one embodiment, the nucleic acid sequence comprises the sequence provided in SEQ ID NO: 1.

[81] The invention further provides nucleic acid encoding the VEGF-binding EV protein of the invention including, e.g., the nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, 11, 13, or 25. In another embodiment, the nucleic acid sequence is shown in SEQ ID NO: 9 or 25.

[82] In yet a further embodiment, the nucleic acid sequence encodes the IL6/IL6 receptor alpha complex-binding EV protein of the invention and the VEGF-binding EV protein of the invention For example, the first nucleic acid sequence encoding the IL6/IL6 receptor alpha complex- binding EV protein may be any of SEQ ID NO: 1, 3, 5, 7, 15, 17, 19, and 21, and the second nucleic acid sequence encoding the VEGF-binding EV protein may be any of SEQ ID NO: 9, 11, 13, or 25.

[83] In another embodiment, the first nucleic acid sequence encoding the IL6/IL6 receptor alpha complex-binding EV protein is any of SEQ ID NO: 1 and 7, and the second nucleic acid sequence encoding the VEGF-binding EV protein selected of any of SEQ ID NO: 9, 11, 13, or 25. [84] In a further embodiment, the first nucleic acid sequence encoding the IL6/IL6 receptor alpha complex-binding EV protein is provided in SEQ ID NO: 1, and the second nucleic acid sequence encoding the VEGF-binding EV protein is any of SEQ ID NO: 9, 1 1 , 13, or 25.

[85] In yet another embodiment, the first nucleic acid sequence encoding the IL6/IL6 receptor alpha complex-binding EV protein Is any of SEQ ID NO: 1, 3, 5, 7, 15, 17, 19, and 21, and the second nucleic acid sequence encoding the VEGF-binding EV protein selected from the group consisting of SEQ ID NO: 9 or 25.

[86] In an additional embodiment, the first nucleic acid sequence encoding the IL6/IL6 receptor alpha complex-binding EV protein is any of SEQ ID NO: 1, 3, 5, 7, 15, 17, 19, and 21, and the second nucleic acid sequence encoding the VEGF-binding EV protein is provided in SEQ ID NO: 25.

[87] In yet a further additional embodiment the nucleic acid sequences comprises a first nucleic acid sequence encoding the IL6/IL6 receptor alpha complex-binding EV protein selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 15, 17, 19, and 21, and a second nucleic acid sequence encoding the VEGF-binding EV protein as provided in SEQ ID NO: 9.

[88] In a specific embodiment, the first nucleic acid sequence encoding the IL6/IL6 receptor alpha complex-binding EV protein is provided in SEQ ID NO: 1, and the second nucleic acid sequence encoding the VEGF-binding EV protein is provided in SEQ ID NO: 25.

[89] In another specific embodiment, the first nucleic acid sequence encoding the IL6/IL6 receptor alpha complex-binding EV protein is provided in SEQ ID NO: 1 , and the second nucleic acid sequence encoding the VEGF-binding EV protein is provided in SEQ ID NO: 9.

[90] Additionally, the invention provides vectors for expressing the IL6/IL6 receptor alpha complex-binding EV protein of the invention which comprises the nucleic acid sequence of the invention.

[91] Further, the invention provides vectors for expressing both the IL6/IL6 receptor alpha complex-binding EV protein of the invention and the VEGF-binding EV protein of the invention, comprising the nucleic acid described herein.

Production of Extracellular Vesicles

[92] Any of the nucleic acids herein can be used in combination for heterologous expression in a cell comprising the IL6/IL6 receptor alpha complex-binding extracellular vesicle (EV) protein and the VEGF-binding EV protein in EV and/or exosome producing cell so as to produce EV and/or exosome. In an embodiment, the nucleic acids are incorporated into vectors for expression in EV and/or exosome producing cells.

[93] Common GMP-grade cells used in such heterologous expression and from which vesicles may be isolated, including extracellular vesicles and exosomes, include HEK293 (human embryonic kidneycell line), variants of HEK293, such as HEK293T, HEK 293-F, HEK 293T, and HEK 293-H, dendritic cells, mesenchymal stem cell (MSCs), HT-1080, PER.C6, HeEa, C127, BHK, Sp2/0, NSO and any variants thereof, and any of the following types of allogeneic stem cell lines: Hematopoietic Stem Cells, such as bone marrow HSC, Mesenchymal Stem Cells, such as bone marrow MSC or placenta MSC, human Embryonic Stem Cells or its more differentiated progeny, such as hESC-derived dendritic cell or hESC-derived oligodendrocyte progenitor cell , Neural Stem Cells (NSCs), endothelial progenitor cells (EPCs), or induced Pluripotent Stem Cells (iPSCs). In an embodiment, any of the cells used for heterologous expression may serve as a source for vesicles, especially extracellular vesicles comprising one or more chimeric vesicle localization moiety(ies) operably linked to GP130 domain 1-3 protein portion or VEGFR-domain fusion units.

[94] For example, the process can be direct engineering of cells for modified vesicles production followed optionally by isolating target modified vesicles subpopulation, i.e., EVs and/or exosomes

[95] Example of engineering cells to produce desired modified vesicles. Vesicle producing cells can be transfected with nucleic acids such as one or more plasmids or viruses carrying nucleic acids encoding the IL6/IL6 receptor alpha complex-binding extracellular vesicle (EV) protein and the VEGF-binding EV protein, either separately or in a single plasmid or virus. The experimental steps can be as the following: a. Culture producer cell line in its optimal growth conditions. b. Prepare the plasmid or virus vector carrying a nucleic acid encoding the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein, either separately or in a single plasmid or virus vector. c. Transfect or infect the vesicle-producing cell lines by the construct made in (b). Transfection can be performed in various ways, such as electroporation or liposome- based nucleic acid transfer. The transfection can be transient or stable transfection.

For establishing a stable IL6/IL6 receptor alpha complex-binding EV protein- and VEGF-binding EV protein-expressing EV -producing cell line, integration of the IL6/IL6 receptor alpha complex-binding EV protein encoding sequence and the

-50- VEGF-binding EV protein-encoding sequence into the recipient cell genome may be needed. In a preferred embodiment, a stable fusion protein-expressing, EV-producing cell line is established wherein a nucleic acid encoding and expressing a fusion protein comprising the GP130 domain 1-3 portion and a vesicle localization moiety, preferably a chimeric vesicle localization moiety, and a nucleic acid encoding and expressing a fusion protein comprising the VEGFR-domain fusion units and a vesicle localization moiety, preferably a chimeric vesicle localization moiety, are integrated into the recipient cell genome, so as to express the fusion proteins which are incorporated into EVs and exosomes and display both GP130 domain 1-3 protein portion and VEGFR-domain fusion units. d. The transfected cell culture is then grown in chemically defined media without FBS for further exosome collection. Alternatively, the transfected cell culture can be seeded into a bioreactor for exosome production. e, Collect the conditioned media after a certain period of time (e.g., 1 day, 2 days, 3 days, 4 days) from regular flask or dish culture or bioreactor culture. f. Isolate modified vesicles from conditioned media. Exosomes may be obtained from the appropriate biological sample using any protocol that yields exosomes useful for therapeutic use, e.g., sufficiently pure, intact exosomes with good stability. The isolation methods can include but are not limited to ultracentrifugation, ultrafiltration, polymer-based pulldown, or immunoaffinity-based pulldown g- Optionally, an antibody, ligand, receptor, and/or aptamer complementary to the fusion proteins displayed on EVs (e.g., anti-GP130 antibody or anti-VEGFR antibody) can be linked to immunomagnetic beads or rods for binding to the EV subpopulation and subsequent isolation. Alternatively, other immune enrichment/isolation techniques can be used. Examples of immunoaffinity capture techniques that may be used to capture exosomes using a selected antibody cocktail include, but are not limited to, immunoprecipitation, column affinity chromatography, magnetic-activated cell sorting, fluorescence-activated cell sorting, adhesion-based sorting and microfluidic- based sorting. The antibodies in the antibody cocktail may be utilized together, in a single solution, or two or more solutions that are used simultaneously or consecutively. [96] In an embodiment, the EV- and/or exosome-producing cell is modified with two separate nucleic acid molecules, each encoding the IL6/1L6 receptor alpha complex-binding EV protein or the VEGF-binding EV protein to obtain EV and/or exosome comprising the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein. Alternatively, the EV- and/or exosome-producing cell may be modified with a single nucleic acid comprising both the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein under the control of a single transcriptional promoter/ enhancer and contained in a single bicistronic mRNA wherein an internal ribosome entry sequence (IRES) separates the two coding sequences and directs expression of the downstream cistron, either the coding sequences for IL6/IL6 receptor alpha complex-binding EV protein or the VEGF-binding EV protein. Bicistronic genes or mini-genes that produce bicistronic mRNAs for the expression of two different proteins are well-known in the art. [97] The invention provides cells for expressing the IL6/IL6 receptor alpha complex- binding EV protein of the invention and the VEGF-binding EV protein of the invention comprising a combination of the vectors described herein, i.e., one that includes the nucleic acid sequence that encodes the IL6/IL6 receptor alpha complex-binding EV protein and another that includes the nucleic acid sequence that encodes the VEGF-binding EV protein. Alternatively, the invention provides cells for expressing the IL6/IL6 receptor alpha complex-binding EV protein of the invention and the VEGF-binding EV protein of the invention comprising a combination of the nucleic acids described herein, i.e., one that encodes the IL6/IL6 receptor alpha complex-binding EV protein and another that encodes the VEGF-binding EV protein. In an embodiment, the cells may comprise the two nucleic acids in separate vectors, each for expressing the IL6/IL6 receptor alpha complex-binding EV protein and the other for expressing the VEGF-binding EV protein. In a separate embodiment, the cells may comprise the two nucleic acids in a single vector producing a bicistronic mRNA comprising sequences encoding the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein separated by an IRES for translation of the downstream cistron (i.e., either the IL6/IL6 receptor alpha complex-binding EV protein-encoding RNA sequence or the VEGF-binding EV protein-encoding RNA sequence).

[98] The invention further provides methods for obtaining the extracellular vesicle or exosome of the invention for inhibiting IL6 trans-signaling and inhibiting VEGF-signaling, comprising culturing an extracellular vesicle and/or exosome-producing cell comprising a combination of the vectors of invention and harvesting culture medium of the extracellular vesicle and/or exosome-producing cell, thereby obtaining the extracellular vesicle and/or exosome of the invention. Alternatively, in a separate embodiment, the methods for obtaining the extracellular vesicle or exosome of the invention for inhibiting IL6 trans-signaling and inhibiting VEGF-signaling comprise culturing an extracellular vesicle and/or exosome-producing cell comprising a single vector comprising a bicistronic mini-gene for expression of a bicistronic mRNA comprising the coding sequences for the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein separated by an IRES for translation of the downstream cistron, and harvesting culture medium of the extracellular vesicle and/or exosome-producing cell, thereby obtaining the extracellular vesicle and/or exosome of the invention.

Pharmaceutical Compositions

[99] Pharmaceutical compositions disclosed herein may comprise modified extracellular vesicles of the invention and/or liposomes with (or without) a payload, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions are in one aspect formulated for intravenous administration or intracranial administration or intranasal administration to the central nervous system. In a preferred embodiment, composition is formulated for intravitreal administration, such as intravitreal injection. Such intravitreal injections may be used to treat eye disease or conditions associated with undesired IL6 trans-signaling resulting in inflammation, oxidative stress, and/or endothelial barrier disruption in human retinal endothelial cells and/or overly active VEGF-signaling resulting in neo-angiogcncsis and vascular permeability. Compositions described herein may include lyophilized EVs (e.g., exosomes). In a preferred embodiment, composition comprises an EV or exosome and a pharmaceutically acceptable excipient.

[100] Pharmaceutical compositions may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[101] Suitable pharmaceutically acceptable excipients are well known to a person skilled in the art. Merely by way of example, excipients include, but are not limited to, surfactants, lipophilic vehicles, hydrophobic vehicles, sodium citrate, calcium carbonate, and dicalcium phosphate. [102] The composition can be formulated into a known form suitable for parenteral administration, for example, injection or infusion. The composition may comprise formulation additives such as a suspending agent, a preservative, a stabilizer and/or a dispersant, and a preservation agent for extending a validity term during storage.

The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient intravitreally, trans arterially, subcutaneously, sublingually, intradermally, intranodally, intramedullary, intramuscularly, intranasally, intraarterially, into an afferent lymph vessel, by intravenous (i.v.) injection, or intracranially injection, or intraperitoneally. In one aspect, the compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, the modified vesicles compositions described herein are administered by i.v. injection. In a different aspect, the modified vesicles compositions described herein are administered by intravitreal injection. Compositions can be administered in a way which allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

Kits Of The Invention

[103] According to another aspect of the invention, kits are provided. Kits according to the invention include package(s) comprising any of the compositions of the invention (including the extracellular vesicles of the invention, fusion proteins that bind IL6/IL6 receptor alpha (i.e., the IL6/IL6 receptor alpha complex-binding EV proteins), fusion proteins that bind VEGF (i.e., the VEGF-binding EV proteins), nucleic acids that encode for the IL6/IL6 receptor alpha complex-binding EV proteins or the VEGF-binding EV proteins, and the vectors that comprise the nucleic acids encoding for the IL6/IL6 receptor alpha complex-binding EV proteins and/or the VEGF-binding EV proteins). In various aspects, the kit comprises any of the compositions of the invention as a unit dose. For purposes herein “unit dose" refers to a discrete amount dispersed in a suitable carrier.

[104] The phrase "package" means any vessel containing compositions presented herein. In preferred embodiments, the package can be a box or wrapping. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes (including pre-filled syringes), bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

[105] The kit can also contain items that are not contained within the package but are attached to the outside of the package, for example, pipettes.

[106] Kits may optionally contain instructions for administering compositions of the present invention to a subject having a condition in need of treatment. Kits may also comprise instructions for approved uses of components of the composition herein by regulatory agencies, such as the United States Food and Drug Administration. Kits may optionally contain labeling or product inserts for the present compositions. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies. The kits can include compositions in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.

[107] The kit may optionally also contain one or more other compositions for use in combination therapies as described herein. In certain embodiments, the package(s) is a container for any of the means for administration such as intravitreal delivery, intraocular delivery, intratumoral delivery, peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular injection, subcutaneous injection, intravenous delivery, intra-arterial delivery, intraventricular delivery, intrasternal delivery, intracranial delivery, or intradermal injection.

[108] Methods of Use

[109] The invention provides methods for inhibiting both IL6 trans-signaling and VEGF signaling in a subject comprising administering an effective amount of a extracellular vesicle and/or exosome of the invention comprising the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein or a pharmaceutical composition of the invention, so as to inhibit both IL6 trans-signaling and VEGF signaling in the subject, thereby inhibiting both IL6 trans-signaling and VEGF signaling in the subject.

[110] The invention further provides methods for reducing both 1L6 trans-signaling and VEGF signaling in a subject comprising administering an effective amount of a extracellular vesicle and/or exosome of the invention comprising the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein or a pharmaceutical composition of the invention, so as to reduce both IL6 trans-signaling and VEGF signaling in the subject, thereby reducing both IL6 trans- signaling and VEGF signaling in the subject. [111] Additionally, the invention provides methods for treating inflammation associated with IL6 trans-signaling and angiogenesis associated with VEGF signaling in a subject comprising administering an effective amount of a extracellular vesicle and/or exosome of the invention comprising the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein or a pharmaceutical composition of the invention, so as to simultaneously inhibit or reduce IL6 trans-signaling and VEGF signaling in the subject, treating inflammation associated with IL6 trans-signaling and angiogenesis associated with VEGF signaling in the subject

[112] In accordance with the practice of the invention, the subject may be afflicted with a tumor, a cancer, a first condition of acute or chronic inflammation due to overactive IL6 trans- signaling, a second condition of angiogenesis or neovascularization from overactive VEGF signaling or excess VEGF level, or a combination thereof.

[113] In some embodiments, the first and/or second condition is any of diabetic retinopathy, retinal vein occlusion, uveitis, choroidal neovascularization, diabetic macular edema, dry eye, dry age-related macular degeneration (AMD), wet AMD, and retinopathy of prematurity.

[114] In some embodiments, the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein are processed or mature proteins lacking a signal peptide.

[115] In some embodiments the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein are formulated as a pharmaceutical composition of the invention.

[116] The invention also provides methods for providing a reservoir of an active agent for treating, inhibiting, or reducing inflammation and angiogenesis in an eye of a subject comprising intravitreal injection of the extracellular vesicle and/or exosome of the invention or a pharmaceutical composition of the invention into the eye of the subject, wherein the extracellular vesicles and/or exosomes reside in the vitreous humor and retina, thereby providing a reservoir of an active agent for treating, inhibiting, or reducing inflammation and angiogenesis in the eye of the subject.

[117] In accordance with the practice of the invention, the subject may be afflicted with a tumor, a cancer, a condition of acute or chronic inflammation due to overactive IL6 trans signaling, a condition of neo-angiogenesis from overactive VEGF signaling or excess VEGF level, or a combination thereof.

[118] In some embodiments, the condition is any of diabetic retinopathy, retinal vein occlusion, uveitis, choroidal neovascularization, diabetic macular edema, dry eye, dry age-related macular degeneration (AMD), wet AMD, and retinopathy of prematurity. [119] The invention further provides methods for treating, inhibiting, or reducing uveitis in an eye of a subject comprising administering an intravitreal injection of the extracellular vesicle and/or exosome of the invention or the pharmaceutical composition of the invention in an effective amount so as to treat, inhibit, or reduce uveitis in the eye of the subject, thereby treating, inhibiting, or reducing uveitis in the eye of the subject.

[120] Additionally provided is a method for treating, inhibiting, or reducing choroidal neovascularization (CNV) in an eye of a subject comprising administering an intravitreal injection of the extracellular vesicle and/or exosome of the invention or the pharmaceutical composition of the invention in an effective amount so as to treat, inhibit, or reduce CNV in the eye of the subject, thereby treating, inhibiting, or reducing CNV in the eye of the subject.

[121] In a specific embodiment of the invention, the CNV is located in the posterior of the eye. Merely as an example, the CNV may be near or around an optic nerve.

. [122] In some embodiments, the treating, inhibiting, or reducing CNV comprises reduction in vascular leakage.

[123] In a specific embodiment, CNV is associated with wet AMD.

[124] In some embodiments of the present disclosure, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In a preferred aspect, the mammal is a human.

[125] Dosages

[126] For purposes of the disclosure, the amount or dose of the active agent (extracellular vesicles of the invention) administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dose of the active agent of the present disclosure should be sufficient to treat cancer as described herein in a period of from about 1 to 4 minutes, 1 to 4 hours or 1 to 4 weeks or longer, e.g., 5 to 20 or more weeks, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular active agent and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated. [127] Many assays for determining an administered dose are known in the art. For purposes herein, an assay, which comprises comparing the extent to which cancer is treated upon administration of a given dose of the active agent of the present disclosure to a mammal among a set of mammals, each set of which is given a different dose of the active agent, could be used to determine a starting dose to be administered to a mammal. The extent to which cancer is treated upon administration of a certain dose can be represented by, for example, the extent of tumor regression achieved with the active agent in a mouse xenograft model. Methods of assaying tumor regression are known in the art and described herein.

[128] The dose of the active agent of the present disclosure also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular active agent of the present disclosure. Typically, the attending physician will decide the dosage of the active agent of the present disclosure with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, active agent of the present disclosure to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the present disclosure, the dose of the active agent of the present disclosure can be about 0.0001 to about 1 g/kg body weight of the subject being treated/day, from about 0.0001 to about 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg body weight/day.

[ 129] Table 1 : Nucleic acid sequences and amino acid sequences

SEQ Sequence Source and note ID NO: 1

-63-

66

76

-90-

AQECSLDDDTILIPI IVGAGLSGLI IVIVIASSHWCCKKEVQETRRER binding EV RRLMSMEMD protein comprises the surface-and- transmembra ne domain of LAMP2b and the cytosolic domain of PTGFRN

SEQ GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN CH3 domain ID YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT of human NO: QKSLSLSPGK * IgGl heavy 49 chain SEQ (GS)3 glycine- ID = GSGSGS serine rich NO: linker 50 SEQ (GS)4 glycine- ID = GSGSGSGS serine rich NO: linker 51 SEQ (GS)₅ glycine- ID = GSGSGSGSGS serine rich NO: linker 52 SEQ (GS)₆ glycine- ID = GSGSGSGSGSGS serine rich NO: linker 53 SEQ (GS)v glycine- ID = GSGSGSGSGSGSGS serine rich NO: linker 54 SEQ (GS)s glycine- ID = GSGSGSGSGSGSGSGS serine rich NO: linker 55 SEQ (GS)9 glycine- ID = GSGSGSGSGSGSGSGSGS serine rich NO: linker 56 SEQ (GS)io glycine- ID = GSGSGSGSGSGSGSGSGSGS serine rich NO: linker 57 92 SEQ (GGS)₂ glycine- ID = GGSGGS serine rich NO: linker 58 SEQ (GGS)3 glycine- ID = GGSGGSGGS serine rich

NO: linker 59

SEQ (GGS)4 glycine- ID = GGSGGSGGSGGS serine rich NO: linker 60 SEQ (GGS)s glycine- ID = GGSGGSGGSGGSGGS serine rich

NO: linker 61 SEQ glycine- ID (GGS)₆ serine rich NO: = GGSGGSGGSGGSGGSGGS linker 62

SEQ (GGGS)₂ glycine- ID = GGGSGGGS serine rich NO: linker 63 SEQ (GGGS)₃ glycine- ID = GGGSGGGSGGGS serine rich

NO: linker 64 SEQ (GGGS)₄ glycine- ID - GGGSGGGSGGGSGGGS serine rich NO: linker 65

SEQ (GGGS)s glycine- ID = GGGSGGGSGGGSGGGSGGGS serine rich NO: linker 66 SEQ (GGGGS)₂ glycine- ID = GGGGSGGGGS serine rich

NO: linker 67 SEQ (GGGGS)₃ glycine- ID = GGGGSGGGGSGGGGS serine rich NO: linker 68

SEQ (GGGGS)₄ glycine- ID = GGGGSGGGGSGGGGSGGGGS serine rich linker NO: 69 SEQ GGGGGS glycine- ID serine rich NO: linker 70 SEQ (GGGGGS)₂ glycine- ID = GGGGGSGGGGGS serine rich NO: linker 71 SEQ (GGGGGS)₃ glycine- ID = GGGGGSGGGGGSGGGGGS serine rich NO: linker 72 SEQ (SG)₂S glycine- ID = SGSGS serine rich NO: linker 73 SEQ (SG)₃S glycine- ID = SGSGSGS serine rich NO: linker 74 SEQ (SG)₄S glycine- ID = SGSGSGSGS serine rich NO: linker 75 SEQ (SG)₅S glycine- ID = SGSGSGSGSGS serine rich NO: linker 76 SEQ (SG)₆S glycine- ID = SGSGSGSGSGSGS serine rich NO: linker 77

SEQ (SG)₇S glycine- ID = SGSGSGSGSGSGSGS serine rich NO: linker 78 SEQ (SG)₈S glycine- ID = SGSGSGSGSGSGSGSGS serine rich NO: linker 79 SEQ (SG)₉S glycine- ID = SGSGSGSGSGSGSGSGSGS serine rich NO: linker 80 SEQ METDTLLLWVLLLWVPGSTGD mouse Ig ID kappa signal NO: peptide from 81 methionine at position 1 to aspartic acid at position 21 of GenBank Accession Number: AAH80787.1

SEQ DYKDDDDK FLAG ID epitope tag NO: 82 SEQ EQKLISEEDL Myc epitope ID tag NO: 83 SEQ YPYDVPDYA HA epitope ID tag NO: 84 SEQ HHHHHH (His)6 epitope ID tag NO: 85

SEQ SDTGRPFVEMYSEI PEI IHMTEGRELVI PCRVTSPNITVTLKKFPLDTLI PD Ig-like

ID GKRI I WDSRKGFI I SNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT I I domain 2 of a

NO: VEGF

86 receptor 1 (VEGFR-1) from serine at position 129 to isoleucine at position 230 of NCBI Reference Sequence Accession Number: NP ,002010.2 or UniProtKB Accession Number: P17948-1 SEQ DVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRD Ig-like ID LKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK domain 3 of a NO: VEGF 87 receptor 2 (VEGFR-2) from aspartic acid at position 225 to lysine at position 327 ofNCBI Reference Sequence Accession Number: NP_002244.1 or UniProtKB Accession Number: P35968-1

SEQ SDTGRPFVEMYSEI PEI IHMTEGRELVIPCRVTSPNITVTLKKFPLDT VEGFR- ID LI PDGKRI IWDSRKGFI ISNATYKEIGLLTCEATVNGHLYKTNYLTHR domain NO: QTNTI I DVVLSPSHGIELSVGEKLVLNCTARTELNVGI DFNWEYPSSK fusion unit 88 HQHKKLVNRDLKTQSGSEMKKFLSTLTI DGVTRSDQGLYTCAASSGLM comprising TKKNSTFVRVHEK Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) from serine at position 129 to isoleucine at position 230 ofNCBI Reference Sequence Accession Number:

NP_002010.2 or UniProtKB Accession Number:

Pl 7948-1 and Ig-like domain 3 of a VEGF receptor 2 (VEGFR-2) from aspartic acid at position 225 to lysine at position 327 ofNCBI Reference Sequence Accession Number: NP_002244.1 or UniProtKB Accession Number: P35968-1

[130] The inventions disclosed herein will be better understood from the experimental details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the inventions as described more fully in the claims which follow thereafter. Unless otherwise indicated, the disclosure is not limited to specific procedures, materials, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[131] EXAMPLES

[132] Example 1: Exosome Production

[133] Engineered exosomes are produced by human cell lines transiently transfected or by stable cell lines expressing the desired vector. Exosomes are then separated from the cells and purified;.

[134] Transient Transfections:

[135] The parental cell line, Expi293F, is used for transient transfections. Cells are grown in Expi293 Expression Medium (ThermoFisher, Cat# Al 435101) and after they reach 3 x IO⁶ cells/ml they are transiently transfected with 1 μg/ml of the selected plasmid DNA(s) using 3.2pl Expifectamine per microgram DNA (ThermoFisher, Cat # A 14524), according to the manufacturer instructions. For production of bispecific exosomes containing two different engineered constructs, vectors for each construct were mixed together at the optimal ratio prior to addition of the Expifectamine reagent. Final plasmid DNA concentration was always maintained at 1 μg DNA per milliliter of culture with 3.2 μl of Expecfactamine reagent added. After 20-24 hours, the media is changed following centrifugation at 300xg for 10 minutes, enhancers are added, and the culture - allowed to grow for 3-4 more days prior to harvest with viability >60%. Cells are separated from exosomes using centrifugation at 300xg for 10 minutes. The supernatant is further centrifuged for 3000xg for 10 minutes and then filtered through a 0.2 μm PES filter. Harvests are stored at -80°C.

[136] Exosome purification

[137] Filtered exosome harvests obtained from the transient transfections are thawed, immediately concentrated 10X and then buffer-exchanged using Tangential Flow-Filtration (TFF1, Omega Filters, PALL) into Phosphate-Buffered Saline (PBS). This intermediate material is further purified by multimodal size-exclusion chromatography on a column (CC700 resin, Cytiva) equilibrated in PBS. The eluate is then further concentrated by a second TFF2 process (Repligen). The purified material is sterile-filtered (0.2 μm PVDF) and formulated with carrier protein

[138] Exosomes Characterization

[139] a) Total exosome concentration by NTA

[140] NTA (Nanoparticle Tracking Analyzer) Nanosight NS300

[141] The technology utilizes the properties of both light scattering and Brownian motion in order to obtain the size distribution and concentration measurement of particles in liquid suspension. A laser beam is passed through the sample chamber, and the particles in suspension in the path of this beam scatter light in such a manner that they can easily be visualized via 20x magnification microscope onto which is mounted a camera. The camera operates at 30 frames per second (fps), capturing a video file of the particles moving under Brownian motion. The software tracks many particles individually and using the Stokes-Einstein equation calculates their hydrodynamic diameters.

[142] Exosomes fall within the particle size range (10 nm to 2000 nm) that can be measured by NTA.

[143] The NanoSight can work with particle concentrations in the range of 10⁷-10⁹ particles/ml, which is approximately 20-100 particles in the field of view. To determine the concentration of exosome preparations in particles per milliliter an aliquot is diluted appropriately to fall within this range and is measured on the NanoSight instrument in two replicates together with a negative and a positive control. The dilution factor is entered into the software by the operator and the output gives detailed size and concentration information for the sample.

[144] b) Qubit - total protein concentration

[145] The Qubit 4 Fluorometer detects fluorescent dyes that bind specifically to the target of interest. This enables precise, accurate measurements even for dilute samples requiring high sensitivity. [146] The total protein concentration of exosome preparations is measured using The Qubit® Protein Assay Kit. The kit includes concentrated assay reagent, dilution buffer, and prediluted BSA standards. The assay reagent is added to an appropriately diluted sample and reacts specifically with proteins. After a 15-minute incubation the standards and the sample are inserted into the instrument one at a time and the output gives a total protein concentration in milligrams per milliliter (mg/mL).

[147] c) Endotoxin measurement - Endosafe Nexgen PTS

[148] The Endosafe® nexgen-PTS™ instrument is aa rapid, point-of-use handheld spectrophotometer that utilizes disposable cartridges for accurate, convenient, and real-time endotoxin, Gram ID, and Beta-glucan testing. Exosome preparation aliquots are appropriately diluted and loaded onto the disposable cartridges in triplicate. The instrument output gives endotoxin concentration in endotoxin units per milliliter (EU/mL).

[149] d) Evaluation of engineering surface protein content on extracellular vesicles (EV) by vesicle flow cytometry analysis.

[150] Cells expressing engineered surface decoy receptors (gp 130 and VEGFR binding sites) were generated to produce exosomes that binds IL-6/IL-6RA complex and VEGF, as a therapeutic modality. Flow cytometric methods were developed to characterize these extracellular vesicles.

[151] Vesicle Flow Cytometry (vFCj for engineered protein detection:

[152] Purified preparations of TEV were assessed by flow cytometry for surface protein levels based on methods developed by Cellarcus Biosciences (La Jolla, CA) for vesicle flow cytometry analysis. In a “no wash” protocol TEV were diluted into vFC Staining buffer (Cellarcus cat no. CBS4), to le9 per mL based on NTA assessment of particles/mL. lx vFRed™ (100X stock, CBS4) was added to the diluted TEV, followed by addition of surface protein antibodies (anti-GP130-PE 1 :20 dilution (BioLcgcnd cat no. 362004), anti-VEGFR-PE 1 :37.5 dilution (Miltenyi cat no. 130-124-438, exploratory constructs with epitope tag anti-FLAG-PE Biolegend 1 :375 dilution Cat no. 637310 (375x)). Samples were incubated at room temperature in the dark for 1 hour. Following incubation, the samples were diluted 1 : 1000 in vFC buffer and run on CytoFlex (Beckman Coulter) at a fixed flow rate 60 μl/min for 2 minutes. CytoFlex was calibrated with Quantibrite for PE quantitation and nanoRainbow beads as a surrogate for diameter originally calibrated to NTA measurements of liposomes. Data was analyzed in FCSExpress by time, excluding the first 30 seconds of collection, the membrane stain height vs. area was used to select vesicle-like events and exclude background. Violet side-scatter height and vFRed™ stain area was used for identification of EVs. This population was then evaluated for expression of surface expressed proteins and data is represented as the % of EV positive for the protein of interest and the mean MESF (molecules of equivalent soluble fluorochrome) of the total EV population as an estimate of the level of protein on the surface of the EVs.

[153] Figure 1 A shows result of vesicle flow cytometry analysis used to determine abundance of gpl 30 on EVs based on the PE-MESF intensity. Cl is a positive control for the anti-GP130 antibody and used for pass/fail criteria in the assay. MB24 contains only VEGFR and does not contain GP130. MB02 and MB06 contain gpl30 proteins and demonstrate measurable levels on their surface. MB02 comprises T102Y, QI 13F, and N1 14L triple mutations in its human GP130 domain 1 which increase binding affinity to IL6/IL6 receptor alpha complex (amino acid residue locations are based on human GP130 sequence as provided in NCBI Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1). See Table 1 and Figure 10 for further details.

[154] Figure IB shows vesicle flow cytometry analysis used to determine abundance of gpl30 on EVs based on the PE-MESF intensity. MB26 contains an empty scaffold with no gpl30 and serves as a negative control. MB16, MB20, MB08 and MB02 contain engineered variations of gpl 30 proteins and demonstrate measurable levels on their surface. All four EV proteins comprise the T102Y, QI 13F, and N114L triple mutations in their human GP130 domain 1 which increase binding affinity to IL6/IL6 receptor alpha complex (amino acid residue locations are based on human GP130 sequence as provided in NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1). Data shown represents epitope tagged versions of the sequences provided in Table 1 (amino terminal FLAG tagged constructs). See Table 1 for further details on the constructs and Figure 10 for schematics of non-epitope tagged constructs (mature or processed constructs lacking signal peptide).

[155] Figure 1C shows vesicle flow cytometry analysis used to determine percent of EVs containing gpl 30 based on the PE fluorescence of anti-gpl 30 antibody. MB26 contains an empty scaffold with no gpl30 and serves as a negative control. MB 16, MB20, MB08 and MB02 contain engineered variations of gp 130 proteins and demonstrate measurable levels on their surface. All four EV proteins comprise the T102Y, QI 13F, and N114L triple mutations in their human GP130 domain 1 which increase binding affinity to IL6/IL6 receptor alpha complex (amino acid residue locations are based on human GP130 sequence as provided in NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1). Data shown represents epitope tagged versions of the sequences provided in Table 1 (amino terminal FLAG tagged constructs). [156] Figure ID shows vesicle flow cytometry analysis used to determine percent of EVs containing chimeric VEGFR based on the PE fluorescence of anti-VEGFR antibody. C2 is an EV that does not contain VEGFR and serves as a negative control. MB24 and MB 10 contain engineered variations of chimeric VEGFR proteins and demonstrate measurable levels of protein on their surface. Data shown represents epitope tagged versions of the sequences provided in Table 1 (amino terminal FLAG tagged constructs). See Table 1 for further details on the constructs and Figure 9 for schematics of non-epitope tagged constructs (mature or processed constructs lacking signal peptide).

[157] Figure IE shows vesicle flow cytometry analysis used to determine percent of EVs containing VEFGR based on the PE fluorescence of anti-VEGFR antibody. C2 is an EV that does not contain VEGFR and serves as a negative control. MB24 and MB 10 contain engineered variations of chimeric VEGFR proteins and demonstrate measurable levels of protein on their surface. Data shown represents epitope tagged versions of the sequences provided in Table 1 (amino terminal FLAG tagged constructs). See Figure 9 for schematics of non-epitope tagged constructs (mature or processed constructs lacking signal peptide).

[158] Example 2: Irt Vitro Functional Assays

[159] Evaluation of signaling effects by EVs with engineered gpl30)/VEGFR vesicles

(EV)

[160] EV displaying engineered proteins that contain portions of VEGFR1 and VEGFR2 binding domains have the potential to antagonize VEGF/VEGFR signaling. In vitro assays were developed to assess the effects of EVs on VEGF signaling. Assays to evaluate the potency of the co- expressed gpl30 binding domain were also developed and the bispecific-EVs were tested in both types of assays.

[161] Assessment of VEGF activation in HUVEC via phospho-VEGFR2 (pVEGFR2)

[162] Primary HUVEC cells (Lonza #CC2519) were plated at 5e4/well in a 96 well plate in a 100 μl volume of complete media (per manufacturer). Cells were incubated at 37°C 5%CO for at least 6 hours to allow for adherence to the plate. Following initial incubation, the media was removed, and the cells were placed in serum free media overnight at 37°C 5%CO₂ . EVs were prepared for dosing by thawing purified EVs on ice, placing 24 pl into highest concentration well and adding BSA to a concentration of 0.25%. 2-fold serial dilutions into PBS w/0.25% BSA were performed for each EV for an 8-point dose response curve. Controls for the assay include buffer only final assay concentration of 0.05% BSA/PBS as a negative control representing no stimulation and establishing background, human VEGF-165 (R&D systems, # 293-VE-500/CF) (20ng/mL final concentration) stimulation alone as a positive control and maximum signal, and a dose response curve of the VEGF inhibitor Aflibercept (ICHOR Bio, #ICH4015) starting at a high concentration of ~2 nM

of 500μg/mL stock ~10 nM). Diluted EVs and aflibercept control dilutions are premixed with VEGF- 165 (20ng/mL) (this is now the cell stimulant) for 1 hour at room temperature prior to adding to the HUVEC cells. Remove the HUVEC cell plate from the incubator and remove the serum free media. Add lOOpl of the stimulant and controls to the cells and incubate 37°C 5%CO₂ for 5 minutes. Immediately remove the media and wash 2X with ice cold PBS. Remove final wash and add 1 10 μl of ice-cold CST lysis buffer (Cell Signaling Technology #9803) containing protease and phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktails, Thermo Scientific #78447). Shake the plate for 15 minutes at 1200 rpm and freeze at -80°C prior to pVEGFR2 (phosphor-VEGFR2) detection. Lysates were analyzed for phospho- VEGFR2 by ELISA (phospho-VEGFR2 ELISA kit, R&D systems, #DYC 1766-5) following manufacturers recommendations. Data was analyzed by GraphPad Prism with IC50 calculated by nonlinear regression analysis 4PL fit with X as particles/mL. [163] Figure 2A shows inhibition of VEGF signaling in HUVEC cells. Briefly, purified preparations of TEV were assessed for their ability to inhibit VEGF ligand in FIUVEC cells measured via phospho-VEGFR2 in the presence of 20 ng/ml VEGF ligand. Monospecific EVs were prepared that contained either MB24 EV protein from EV -producing cells transfected with a vector for expressing MB24 cDNA (SEQ ID NO: 23) or MB 10 EV protein from EV-producing cells transfected with a vector for expressing MB 10 cDNA (SEQ ID NO: 9). EVs were prepared that contained both MB10/MB02 EV proteins from EV-producing cells co-transfected with a vector for expressing MB 10 cDNA (SEQ ID NO: 9) and a vector for expressing MB02 cDNA (SEQ ID NO: 1). Similarly, EVs were prepared that contained both MB24/MB02 EV proteins from EV-producing cells co-transfected with a vector for expressing MB24 cDNA (SEQ ID NO: 23) and a vector for expressing MB02 cDNA (SEQ ID NO: 1). Additionally, EVs were prepared that contained both MB10/08 EV proteins from EV-producing cells co-transfected with a vector for expressing MB 10 cDNA (SEQ ID NO: 9) and a vector for expressing MB 08 cDNA (SEQ ID NO: 7). EVs were prepared also that contained both MB24/MB08 EV proteins from EV-producing cells co-transfected with a vector for expressing MB24 cDNA (SEQ ID NO: 23) and a vector for expressing MB08 cDNA (SEQ ID NO: 7). Monospecific EV preparations and EV preparations were analyzed for their ability to inhibit VEGF signaling in HUVEC. IC50 values are provided within parentheses for the monospecific MB24 EV (closed squares, IC50 of 1.91E+09 EV/mL) and monospecific MB10 EV (closed circles IC50 of 1.07E+09 EV/mL) which are monospecific VEGF inhibitors comprising VEGFR-domain fusion units. IC50 values are provided within parentheses for the bispecific MB10/MB02 EVs (open circles, of 1.95E+09

EV/mL), bispecific MB10/MB08 EVs (closed triangles, IC50 of 6.88E+09 EV/mL), bispecific MB24/MB02 EVs (open squares, IC50 of 1.87E+10 EV/mL) and bispecific MB24/MB08 (closed inverted triangles, IC50 of 7.90E+10 EV/mL). MB02 and MB08 are gp!30 constructs that inhibit 1L6- trans-signaling. All EVs containing VEGFR decoy receptors inhibited VEGF ligand signaling with varying potency. Data shown represents epitope tagged versions of the sequences provided in Table 1 (amino terminal FLAG tagged constructs) and schematically represented in Figures 9 and 10 without the epitope tag. Representative data shown. P/mL - EV particles/mL.

[164] Assessment of VEGF inhibition asins a VEGF-activated reporter assay (Promeea)

[165] To measure the potency of EVs engineered to block VEGF ligand signaling, we utilized Promega’s VEGF Bioassay (Catalog number: GA2001 ) according to the manufacturer’s recommendations. The VEGF Bioassay kit contains a single-use vial of frozen cells engineered to express human VEGF receptor 2 coupled to a luciferase-based reporter readout. To test the inhibitory activity of crude exosomes (filtered culture supernatant harvests) or purified EV preparations, EV samples were serially diluted two-fold in 50μl of complete tissue culture media containing 15 ng/ml VEGF-165 ligand (Promega Cat# J237A) and incubated for 45 min at room temp. Freshly thawed cells (KDR/NFAT-RE HEK293) were then added (25 μl 40,000/well) and allowed to incubate for six hours at 37°C in a CO₂ (5% ) incubator in a final volume of 75pl. The VEGF-dependent induction of luciferase in the assay cells was then determined by adding 75 pl of the luciferase detection reagent (Bio-Gio™ Luciferase Assay System, catalog#: G7940), allowed to mix for ten minutes. The solubilized cell assay samples were then transferred to a solid white assay plate (Thermo Fisher Scientific cat#: 9502887) and the luminescence determined on an Agilent BioTek Synergy Hl plate reader. The dilution of EVs at which 50% of the VEGF activity was inhibited was defined as one unit of inhibitory activity.

[166] Figure 2B is an analysis of purified preparations of TEV which were assessed for their ability to inhibit VEGF ligand activity using Promega’s VEGF Reporter assay in the presence of 10 ng/ml VEGF ligand. EV preparations that contain MB 10 EV protein produced from cells transfected with MB 10 coding sequences (SEQ ID NO: 9) as a monospecific anti-VEGF inhibitor or a bispecific combination of MB 10 EV protein with MB02 EV protein, a GP130 construct that inhibits IL6-trans- signaling, produced from cells co-transfected with both MB 10 coding sequences (SEQ ID NO: 9) and MB02 sequences (SEQ ID NO:1) were compared. Both the monospecific (MB10) and the bispecific (MB10/MB2) EV preparations were effective at inhibiting VEGF signaling. The monospecific EVs (MB10, solid circles) had an EC50 value of 1.08E+09 EVs/ml while the EVs (MB10/MB2, open circles) had an EC50 value of 2.81E+09 EVs/ml. All constructs expressed in the EVs used in this experiment contain an N-terminal FLAG epitope tag sequence. See Table 1 and Figures 9 and 10 for details of MB02 and MB 10 EV proteins without the N-terminal FLAG epitope tag found between the signal peptide sequence and the GP130 domain 1-3 protein portion or VEGFR-domain fusion units.

[167] Evaluation of signaling effects by EVs with engineered gp130/VEGFR vesicles

(EV)

[168] EV displaying engineered proteins that contain domains of gpl30 have the potential to antagonize IL-6 classic and trans-signaling. In vitro assays were developed to assess the effects of EVs on IL-6 trans-signaling. An assay in which primary human cells were stimulated with a complex of human IL-6/soluble IL-6Ra resulting in phospho-STAT3 was utilized to assess trans-signaling.

[169] Assessment of IL-6 trans-signaling activation in HUVEC via phospho-STAT3

[170] Primary HUVEC cells (Lonza #CC2519) were plated at 1.5e4/well in a 96 well plate in a 100 pl volume of complete media (per manufacturer). Cells were incubated at 37°C 5%CO₂ for 6 hours to allow for adherence to the plate. Following initial incubation, the media was removed, and the cells were placed in serum free media overnight at 37°C 5%COz. Stimulation of IL-6 trans- signaling requires generation of a complex between human IL-6 (PeproTech #50-399-370) and soluble human IL-6Ra (PeproTech #50-162-4794). IL-6 and IL-6Ra were combined at a 1 : 1 ratio at a concentration of 8.5 nM each (6x) to allow for complex formation at room temperature for 30 minutes. EVs were prepared for dosing by thawing purified EVs on ice, placing 24 μl into highest concentration well and adding BSA to a concentration of 0.25%. 3-fold serial dilutions into PBS w/0.25% BSA were performed for each EV for an 8-point dose response curve. Controls for the assay include buffer only final assay concentration of 0.05% BSA/PBS as negative control representing no stimulation and establishing background, human IL-6/IL-6Ra complex (1.4 nM final concentration) stimulation alone as a positive control and maximum signal, and a 3-fold serial dilution dose response curve of the human sgpl30-Fc (R&D systems. 671-GP-MTO) starting at a high concentration of 250 nM. Diluted EVs and gpl30-Fc control dilutions are premixed with IL-6/IL-6Ra complex (1.4 nM) (this is now the cell stimulant) for 1 hour at room temperature prior to adding to the FIUVEC cells. Remove the HUVEC cell plate from the incubator and remove the serum free media. Add lOOpl of the stimulant and controls to the cells and incubate 37°C 5%CO₂ for 15 minutes. Immediately remove the media and wash 2X with ice cold PBS. Remove final wash and add 1 10 pl of ice-cold CST lysis buffer (Cell Signaling Technology #9803) containing protease and phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktails, Thermo Scientific #78447). Shake the plate for 15 minutes at 1200 rpm and freeze at -80°C prior to phosphor-STAT3 (pSTAT3) and total STAT3 detection. Lysates were analyzed for total STAT3 (Cell Signaling Technologies PathScan® Total Stat3 Sandwich ELISA Kit #7305) and phosphor-STAT3 ( Cell Signaling Technologies PathScan® Phospho-STAT3 (Tyr705) Chemiluminescent Sandwich ELISA Kit #7149) following manufacturers recommendations. 50ul of lysate per sample was assessed for pSTAT3 and 30 μl for total STAT3. pSTAT3 was normalized to total STAT3 and analyzed by GraphPad Prism with IC50 calculated by nonlinear regression analysis 4PL fit with X as EV particles/mL.

[171] Figure 2C shows inhibition of IL-6 trans-signaling in HUVEC cells by mono-specific IL6 trans-signaling inhibitors. Purified preparations of TEV were assessed for their ability to inhibit IL-6/IL-6RA ligand in HUVEC cells measured via phospho-STAT3. EV preparations that contain MB08 (closed triangles, IC50 of 2.08E+11 EV/mL) and MB02 (closed circles IC50 of 7.38E+10 EV/mL) as monospecific IL-6 trans-signaling inhibitors. All EVs containing gpl30 constructs inhibited IL-6/IL-6Ra ligand signaling with varying potency. See Table 1 and Figure 9 for details of MB02 and MB08 proteins. Representative data shown.

[172] Figure 2D shows inhibition of IL-6 trans-signaling in HUVEC cells by monospecific IL-6 trans-signaling inhibitor and bispecific IL-6 trans-signaling and VEGF-signaling inhibitor. Purified preparations of TEV were assessed for their ability to inhibit IL-6/IL-6RA ligand in FIUVEC cells measured via phospho-STAT3. EV preparations that contain MB02 (closed triangles, IC50 of 1.43E+10 EV/mL) as a monospecific IL-6 trans-signaling inhibitor or bispecific combinations of MB10/MB02 (open circles, IC50 of 3.22E+10 EV/mL) and MB24/MB02 (open squares, IC50 of 1.73E+10 EV/mL ). MB10 and MB24 are VEGF decoy receptor constructs that inhibit VEGF signaling. All EVs containing gpl30 constructs inhibited IL-6/IL-6RA ligand trans-signaling with varying potency. Data shown represents epitope tagged versions of the sequences provided (amino terminal FLAG tagged constructs). Representative data shown.

[173] Assessment of inhibition ofIL6 trans-signaling using In Vivogen ’s IL6 reporter assay

[ 174] To measure the potency of EVs for blocking IL6-trans-signaling, we used Invivogen’s

IL6 reporter assay (cat#: hkb-hil6) to detect the activity of 0.1 nM human hyper-IL6 (R&D Systems cat#: 8954-SR), a fusion protein of human IL6 receptor alpha coupled to IL6 with a linker. HEK-Blue cells were grown to greater than 70% confluency in complete DMEM media (Cat#: Corning- 10-013- CM ) containing 10% FBS (Gibco Cat #A4766801), IX Penn/Strep (cat #SIG-P0781-100ML), and lx selection reagent (cat # hb-sel). The day before the assay was to be run the HEK-Blue cells were lifted off the tissue culture plate using phosphate buffered saline containing lOmM EDTA, counted for cell density, then plated out in lOOμl of complete media at 100,000 cells per well. 18-24 hours later EV samples, either crude exosomes (filtered culture supernatant harvests) or purified EV preparations, were serially diluted in a separate dilution plate in 130ql of complete tissue culture media (2X the final EV concentration) containing 0.2 nM Hyper-IL6. After incubating the dilution plate for 45 min, l OO μl was then transferred into the HEK-Blue cell plate to begin the reporter assay at a final assay concentration of IX for EV samples and 0.1 nM hyper-IL6. 20-24 hours later 20 μl of assay culture supernatant was transferred to 180 μl of Quanti-Blue Solution (cat#rep-qbs2) in a separate plate and allowed to incubate at 37°C for 1 -2 hours. To detect the total amount of SEAT secreted from the HEK-

Blue cells the absorbance was read at 655nm on an Agilent BioTek Synergy Hl plate reader. The dilution of EVs at which 50% of the Hyper-IL6 activity was inhibited was defined as one unit of IL6- TS inhibitory activity.

[175] Figure 2E shows analysis of purified preparations of TEV for their ability to inhibit

IL6-trans-signaling using InvivoGen’s HEK-Blue reporter assay stimulated with 0.1 nM hyper-IL6. Monospecific EVs for inhibiting IL6 trans-signaling were obtained from cells transfected with a vector for the expression of MB02 cDNA (SEQ ID NO: 1), which encodes an IL6/IL6 receptor alpha complex-binding EV protein (see Figure 10). EVs for inhibiting both IL6-trans-signaling and VEGF- signaling were obtained from cells co-transfected with both a vector comprising MB02 cDNA (SEQ ID NO: 1) to express an IL6/IL6 receptor alpha complex-binding EV protein (see Fgiure 10) and a vector comprising MB 10 cDNA (SEQ ID NO: 9) to express a VEGF-binding EV protein with three VEGFR-domain fusion units (see Figures 9). Both the monospecific (MB2) and the bispecific (MB2/MB 10) EV preparations were effective at inhibiting IL6 trans-signaling. The monospecific EVs (MB2, solid triangles) had an EC50 value of 6.23E+08 EVs/ml while the EVs (MB2/MB10, open triangles) had an EC50 value of 8.22E+08 EVs/ml.

[176] Example 3: VEGFR-Binding Domain Concatemers

[177] Construction and evaluation of VEGFR-binding domain concatemers

[178] To enhance the affinity and potency of our bispecific exosomes, several concatenated protein constructs were designed. Concatenated protein constructs involve the back-to-back repetition of a single protein entity that alone is capable of binding to a specific target but when repeated in duplicate or triplicate demonstrates increased binding potential and increased binding capacity for the target representing a new and unique protein structure. The single protein entity that is to be repeated can be composed of one protein domain, or multiple domains combined that represent a single binding region. To concatenate a single protein entity, the coding sequence for the C-terminus of the first protein entity is joined to the coding sequence for the N-terminus of the second protein entity. To optimize the correct protein folding of each protein region in the concatemer, the protein regions will typically be separated by a flexible linker of between 8-16 amino acids. However, direct coupling without the presence of a linker may be preferred. The resulting nucleotide coding sequence when expressed as a single protein will represent a new protein with unique features or benefits compared to existing non-concatenated protein structures.

[179] To generate a concatenated protein structure containing two VEGF ligand binding domains repeated in duplicate (SEQ ID NO: 12), the single protein entity represented by domain 2 of VEGFR1 (NP-002010.2, AA 129-230) and domain 3 of VEGFR2 (NP_002244.1 , AA 225-327) was repeated two times using a nine amino acid linker of (SG)4S to separate both VEGF binding regions. See Table 1, SEQ ID NOs: 11 and 12 for descriptions.

[ISO] To generate a concatenated protein structure containing three VEGF ligand binding domains repeated in triplicate (SEQ ID NO: 10), the single protein entity represented by domain 2 of VEGFR1 (NP_002010.2, AA 129-230) and domain 3 of VEGFR2 (NP_002244.1 , AA 225-327) was repeated three times using a nine amino acid linker of (SG)4S to separate all three VEGF binding regions

[181] Concatenated designs for VEGFR1/R2 binding sites are described below:

[182] Version 1 TEV: (SEQ ID NO: 23)

[183] Coding sequence for MB24 EV protein comprising mouse Ig kappa signal peptide

(AAH80787.1, AA 1-21), then VEGF receptor 1 domain 2 (Accession number NP 002010.2, AA 129-230) then VEGF receptor 2 domain 3 (Accession number NP_002244.1, AA 225-327) followed by a CH3 domain (Accession number AAL96263.1, AA 110-216) then LAMP2 isoform b (Accession number NP_054701.1, AA 29-395) surface-and-transmembrane domain and PTGFRN cytosolic domain (Accession number NP 065173.2, AA 854-879) from vector 780; Artificial Sequence.

[184] Version 2 TEV: (SEQ ID NO: 9)

[185] Coding sequence for MB 10 EV protein comprising mouse Ig kappa signal peptide (AA1180787.1 , AA 1 -21 ), then VEGF receptor 1 domain 2 (Accession number NP_002010.2, AA 129-230) then VEGF receptor 2 domain 3 (Accession number NP_002244.1, AA 225-327) followed by a second copy of VEGF receptor 1 domain 2 and VEGF receptor 2 domain 3 , followed by a third copy of VEGF receptor 1 domain 2 and VEGF receptor 2 domain 3, then followed by a CH3 domain (Accession number AAL96263.1, AA 110-216) then LAMP2 isoform b (Accession number NP_054701.1, AA 29-395) surface-and-transmembrane domain and PTGFRN cytosolic domain (Accession number NP_065173.2, AA 854-879) from vector 779; Artificial Sequence.

[186] Monospecific TEVs (M-TEVs) that bind and block VEGF ligand were produced from either a vector that expressed a single VEGFR-domain fusion unit (SEQ ID NO: 23) or a vector that expressed a triple concatenated VEGFR-domain fusion units (SEQ ID NO: 9), and the monospecific TEV with VEGFR1/2 decoy receptors comprising either single or triple concatenated VEGFR-domain fusion units were isolated as described in Example 1. Bispecific TEVs (B-TEVs) that bind both IL6/IL6 receptor alpha complex and VEGF to inhibit IL6 trans-signaling and VEGF-signaling, respectively, were produced by co-transfecting two vectors, each expressing SEQ ID NO: 23 or 9 and other expressing SEQ ID NO: 1, to obtain B-TEVs comprising a VEGF-binding EV protein with a single or triple concatenated VEGFR-domain fusion units (i.e., MB24 or MB 10) and an IL6/IL6 receptor alpha complex-binding EV protein (i.e., MB2), respectively. The EVs were then evaluated for potency in the HUVEC phospho- VEGFR2 in vitro assay as described in Example 2.

[187] Figure 3 A shows that M-TEVs comprising a VEGF-binding EV protein having a single VEGFR-domain fusion unit inhibits VEGF signaling (MB24 IC50 of 1 .91E+09 EV/mL, solid squares) to a similar extent as M-TEVs comprising a VEGF-binding EV protein having triple concatenated VEGFR-domain fusion units (MB 10 IC50 of 1.07E+09 EV/mL, solid circles). When the same VEGF- binding EV protein (either MB24 or MB 10) is co-expressed with an IL6/IL6 receptor alpha complex- binding EV protein (i.e., MB2) to produce B-TEV with both VEGF-binding EV protein and IL6/IL6 receptor alpha complex-binding EV protein, the B-TEV comprising the VEGF-binding EV protein with a single or non-concatenated VEGFR-domain fusion unit had reduced potency by about a factor of 5-10 fold (MB24/MB02 IC50 of 1.87E+10 EV/mL, open squares) as can be seen in Figure 3B. In contrast, the B-TEV comprising the VEGF-binding EV protein with triple concatenated VEGFR- domain fusion units (i.e., MB 10) and the same IL6/IL6 receptor alpha complex-binding EV protein (i.e., MB02) had a similar inhibitory or slight decrease (~ 2-3x) in inhibitory activity (MB10/MB02 IC50 of 1.95E+09 EV/mL, open circles in Figure 4B) as the M-TEV comprising the VEGF-binding EV protein with triple concatenated VEGFR-domain fusion units. An orthogonal measure of construct abundance on the surface of EVs called by vesicle flow cytometry (vFC) confirmed the reduction in potency seen here. The reduction in abundance and potency was unique to the non-concatenated construct and only seen when produced in a B-TEV format. Data shown in Figure 3 A and B represents epitope tagged versions of the sequences provided in Table 1 (amino terminal FLAG tagged constructs) and are representative results.

[188] Example 4: vFC for evaluation of bispecific expression

[189] Evaluation of engineering surface protein content on extracellular vesicles (EV) by vesicle flow cytometry analysis

[190] Extracellular vesicles are released by cells under normal physiological and pathological conditions. They are surrounded by a lipid bilayer which contains proteins derived from the cells they originated from and can carry additional molecules including DNA and RNA. Cells expressing more than one engineered surface receptor (gpl30 and VEGFR) were generated to produce exosomes as a therapeutic modality. Flow cytometric methods developed to characterize extracellular vesicles were used to assess the presence and abundance of each protein relative on our TEV modalities.

[191] Vesicle Flow Cytometry (vFC) for engineered protein detection:

[192] Purified preparations of TEV were assessed by flow cytometry for surface protein levels based on methods developed by Cellarcus Biosciences (La Jolla, CA) for vesicle flow cytometry analysis. In a “no wash” protocol, TEV were diluted into vFC Staining buffer (Cellarcus cat no. CBS4), to le9 per mL based on NTA assessment of particles/mL. lx vFRed™ (100X stock, CBS4) was added to the diluted TEV, followed by sequential addition of protein detection antibodies (anti- GP130-PE 1 :20 dilution (BioLegend cat no. 362004), anti-VEGFR-APC 1 :80 dilution (Miltenyi Cat no. 130-120-706)) Samples were incubated at room temperature in the dark for 1 hour. Following incubation, the samples were diluted 1 :1000 in vFC buffer and run on CytoFlex (Beckman Coulter) at a fixed flow rate 60μl/min for 2 minutes. CytoFlex was calibrated with Quantibrite for PE and nanoRainbow beads as a surrogate for diameter originally calibrated to NTA measurements of liposomes. Data was analyzed in FCSExpress by time, excluding the first 30 seconds of collection, the membrane stain height vs area was used to select vesicle like events and exclude background. Violet side scatter height and vFRed™ stain area was used for identification of EVs. This population was then evaluated for dual expression of surface expressed protein and data is represented as the % of EV positive for both proteins of interest.

[193] Figure 4 shows vesicle flow cytometry analysis to determine abundance of gpl30 (PE- A) and VEGFR (APC-A) on EVs comprising MB10/MB02 EV proteins. Panel (a) shows a dot plot identifying EVs with both MB10/MB02 EV proteins on the surface showing percent of dual stained population (boxed area). Panels (b) and (c) show histograms of an estimate of the number of the IL6/IL6 receptor alpha complex-binding EV protein per EV based on MESF determination with PE- A labeled anti-GP130 antibody (PE- A (MESF)) and individual mean fluorescence intensity for VEGFR1/2R (APC-C), respectively. Data shown represents epitope tagged versions of the sequences provided in Table 1 (amino terminal FLAG tagged constructs). Representative data shown. PE-A has been calibrated to determine an estimate of the number of sites per EV. The APC channel is not calibrated, and this represents relative levels - not quantitative

[194] EVs were produced and isolated as described in Example 1 from cells engineered to co-express both VEGFR1/2 and gpl30 constructs (i.e., MB 10/MB02 EV proteins). Flow cytometry was employed to determine the abundance of both constructs being present on the same EV demonstrated by dual stained vesicles. Figure 4, panel (a) shows that 75% of the EVs co-expressed the VEGF-binding EV protein and IL6/IL6 receptor alpha complex-binding EV protein.

[195] Example 6: In Vivo EV Biodistrihution

[196] EVs are a new therapeutic modality for which understanding the biodistribution in the ocular space will be important. The route of administration for the target indications will be intravitreal (IVT) injection. A surrogate EV was developed for evaluation of biodistribution. A NanoLuc construct designed to enrich NanoLuc in the EVs was overexpressed in the producer cell line. EVs were purified from the supernatant following the methods described. To determine the biodistribution of the EV, rabbits were dosed IVT with 50μl of EV at a concentration of up to le! 3/mL. 24 hours after dosing the animals were sacrificed and ocular tissues were collected. Aqueous humor and vitreous were collected prior to enucleation of the eye. After enucleation the eye was dissected to collect the cornea, lens, ICB, retina, choroid and sclera. All tissues were weighed, snap frozen in liquid nitrogen and stored at -80 prior to processing. Tissues were homogenized in IX RIPA buffer with protease and phosphatase inhibitors. Total protein was determined by BCA analysis and NanoLuc was measured following manufacturers recommendations for Promega Nano-Gio Assay System and read on BioTek Synergy for luminescence. A standard curve was generated using the same TEV that was dosed in vivo to determine luminescence per particle based on NTA as described. Data was represented as TEV particlcs/tissue weight in grams.

[197] Figure 5 shows biodistribution of TEV following single injection in rabbit eye. Purified preparations of TEV were dosed IVT into rabbits and ocular tissues were harvested 24 hours post injection. Tissues were homogenized in RIPA buffer with proteases and phosphatases and NanoLuc activity was assessed. n=3 rabbits dosed and tissues analyzed. Data shown represented as the number of TEV particles/ gram of tissue. ICB - iris, ciliary body

[198] A majority of the TEV dose was detected in the vitreous humor 24 hours after IVT dosing. This aligned with it being retained in the vitreous where it was dosed and the TEVs arc not rapidly cleared from the site of injection. Retina had the second highest amount of TEVs. The anterior tissues of the eye (aqueous humor, cornea, ICB and lens) had low to no detectable levels of TEV indicating that at 24 hours the TEV remained mainly in the posterior of the eye.

[199] Example 7; In vivo Rat Choroidal Neovascularization (CNV) Model

[200] EV as a therapeutic modality has potential to be beneficial for treatment of diseases in the posterior of the eye. Choroidal neovascularization (CNV) can cause significant loss of vision and potential blindness and is a pathology associated with several ocular diseases including diabetic retinopathy. Animal models of CNV have been developed to characterize CNV formation and for the evaluation of potential therapeutics.

[201] CNV was modeled in Brown Norway rats using an 810nm diode laser to create four lesions per eye around the optic nerve. TEVs or controls were introduced by IVT following the laser induction. Fundus imaging was performed on day 1 post induction to document lesion formation and exclude any lesions that resulted in hemorrhage or did not disrupt Bruch’s membrane. Fluorescein angiography (FA) was performed on day 8 and day 15 to measure vascular leakage at the lesion sites. Fluorescein was injected IP and measurements were taken approximately 1 minute following injection. TEVs were also dosed on by IVT on day 8 following FA imaging.

[202] Figure 6 shows effect of B-TEV or Eylea treatment on lesion area in rat 15 days post laser induced CNV measured by fluorescien angiography. Purified preparations of TEV were dosed IVT on day 1 and day 8 into rat eyes following laser induced CNV. Vehicle (closed circles) shows robust lesion formation that is decreased by Eylea 200μg (closed triangles). B-TEV (1.28E12/mL) with gpl30 and VEGFR (plus symbol +) also decreased lesion area. Lesion area was assessed by fluorescein angiography on day 15. Data is represented by the lesion area measured in pixels shown as the mean +/- SEM. Each data point represents the mean of lesion measurements per eye. Ordinary one-way ANOVA (GraphPad Prism) was applied to the data sets compared to the vehicle control group. **** p<0.0001

[203] Laser-induced choroidal neovascularization (CNV) was performed on Brown Norway rats on Day 1 in all groups. Lesions were measured on day 15 for vascular leakage by fluorescein angiography (FA). Both the Eylea and B-TEV treated groups had significant reduction in the size of the lesions compared to the vehicle control.

[204] Example 8: In Vivo Mouse Endotoxin-Induced Uveitis (EIU) Model

[205] B-TEV containing an IL6/IL6 receptor alpha complex-binding EV protein and a VEGF-binding EV protein was evaluated for its ability to inhibit IL-6/IL-6Ra complex trans-signaling through cell-surface bound gp!30 co-receptor. Endotoxin induced uveitis in rodents demonstrate increased levels of IL-6 in response to EPS, dosed either systemically or IVT. B-TEV was evaluated in a mouse model of EIU where B-TEV was dosed IVT followed by EPS (5mg/kg) dosing IP. 18 hrs post EPS treatment, eyes were enucleated and dissected into anterior and posterior tissues for evaluation of response. IL-6/IL-6R trans-signaling through gpl30 results in STAT3 being phosphorylated at the start of the signaling cascade. pSTAT3 (phospho-STAT3) was measured in ocular lysates by ELISA and normalized to total STAT3 also measured by ELISA

[206] Figure 7 shows effects of B-TEV on pSTAT3 induction in mouse endotoxin induced uveitis model. Purified preparations of B-TEV were dosed IVT into mice followed by EPS challenge administered IP. Eyes were enucleated 18 hours post EPS administration. Tissues were dissected into the anterior or posterior portion of the eye and homogenized in RIP A buffer with proteases and phosphatases. Groups tested included a PBS control group (PBS IVT/PBS IP; closed circles), B-TEV control group (B-TEV IVT/PBS IP; closed triangles), EPS induction group (PBS IVT/LPS IP; closed squares) and B-TEV treatment group (B-TEV IVT/LPS IP; open circles). pSTAT3 and total STAT3 were analyzed by ELISA (CST). Data represented as the ratio of pSTAT3 to total STAT3.

[207] Systemic EPS induced pSTAT3 in anterior ocular tissue of mice 18 hrs post challenge. TEVs alone did not induced pSTAl’3. TEVs with gpl30 dosed IVT reduced the pSTAT3 response to systemic EPS,

[208] Example 9: Exemplary Expression Vector for Production of IL6/IL6 Receptor Alpha Complex-Biriding EV Protein and VEGF-Binding EV Protein of the Invention Incorporated Into an EV and/or Exosome

[209] Figure 8 shows an exemplary plasmid map or vector, MB04 protein expression vector, used to produce MB04 EV protein in mammalian cells comprising an IL6/IL6 receptor alpha complex-binding extracellular vesicle (EV) protein. A CMV enhancer/promoter drives the expression of MB04 cDNA (SEQ ID NO: 3), while the SV40 poly(A) signal at the 3’end of the MB04 cDNA cassette permits polyadenylation of the resulting MB04 RNA. For producing mammalian cells stably transfected with vMB04 expression vector, a puromycin-resistant minigene is present on the same plasmid and may be used to select for puromycin resistance following transfection of the vMB04 DNA. The vMB04 expression vector can be used to express other IL6/IL6 receptor alpha complex-binding extracellular vesicle (EV) proteins or a VEGF-binding EV protein by replacing the MB04 cDNA sequence (SEQ ID NO: 3) with the desired cDNA sequences, such as, for example, MB02 cDNA sequence (SEQ ID NO: 1), MB06 cDNA sequence (SEQ ID NO: 5), MB08 cDNA sequence (SEQ ID NO: 7), MB10 cDNA sequence (SEQ ID NO: 9), MB12 cDNA sequence (SEQ ID NO: 11), MB14 cDNA sequence (SEQ ID NO: 13), MB16 cDNA sequence (SEQ ID NO: 15), MB 18 cDNA sequence (SEQ ID NO: 17), MB20 cDNA sequence (SEQ ID NO: 19), MB22 cDNA sequence (SEQ ID NO: 21), MB24 cDNA sequence (SEQ ID NO: 23), or MB25 cDNA sequence (SEQ ID NO: 25).

[210] Figure 9 provides a schematic of representative VEGF-binding EV proteins anchored to a surface of an extracellular vesicle or exosome. The VEGFR-domain fusion units are N-terminal to the chimeric vesicle localization moiety and the external or outer surface location of the VEGFR- domain fusion units and the surface domain of the chimeric vesicle localization moiety. The mature or processed VEGF-binding EV proteins (shown here) lack a N-terminal signal peptide, necessary for insertion and passage of the N-terminal portion of the newly translated or nascent type I transmembrane proteins through the lipid bilayer. The transmembrane (TM) domain of the chimeric vesicle localization moiety resides in the lipid bilayer and anchors the VEGF-binding EV protein at the surface of the EV or exosome. The cytosolic domain (shown as a tail and labeled PTGFRN) resides in the interior of the EV or exosome. Flexible linkers rich in glycine and serine residues may be present, separating different functional portions of the VEGF-binding EV protein. An immunoglobulin heavy chain constant region 3 (CH3) may be present between the VEGFR-domain fusion units and the chimeric vesicle localization moiety. The chimeric vesicle localization moiety shown here comprises the surface-and-transmembrane domain of Lamp2B and its cytosolic domain replaced with the cytosolic domain of PTGFRN. Lastly, an epitope tag may be present in the mature or processed VEGF-binding EV proteins either at the N-terminus or C-terminus. When present in the VEGF-binding EV proteins used in the examples, the epitope tag is present in the N-terminus. Prior to incorporation into an EV or exosome, the N-terminally located epitope tag may be found between the signal peptide and the most N-terminal VEGFR-domain fusion unit.

[211] Figure 10 provides a schematic of representative IL6/IL6 receptor alpha-binding EV proteins anchored to a surface of an extracellular vesicle or exosome. The IL6/IL6 receptor alpha- binding EV protein comprises a mammalian (preferably human) GP130 domain 1-3 (D1 -D3) protein portion of a full length GP130 protein and a C-terminal chimeric vesicle localization moiety. The GP130 domain 1-3 protein portion may extend in the C-terminal direction to include additional domains, resulting in IL6/IL6 receptor alpha-binding EV proteins comprising GP130 domains 1-4 protein portion, GP130 domains 1-5 protein portion, or GP130 domains 1-6 protein portion arranged in an order as would be found in a fully length native GP130 protein The GP130 domain 1 (DI) may have additional amino acid residue changes at a conserved glutamine-asparagine dipeptide and a threonine, arginine, or glutamine eleven amino acids upstream of the conserved glutamine- asparagine dipeptide, wherein the glutamine or asparagine of the dipeptide may be changed to phenylalanine or leucine and the upstream threonine, arginine, or glutamine may be changed to a tyrosine, singly or in combination, to increase the binding affinity of the GP130 protein portion to IL6/IL6 receptor alpha complex. In a preferred embodiment, all three amino acids are changed, such that for example, human GP130, domain 1 comprises amino acid substitutions, T102Y, QI 13F, and N 1 14L, based on human GP130 amino acid sequence as provided in NCBI Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1 (indicated as YFL in DI of Figure 10). While the newly translated or nascent IL6/IL6 receptor alpha complex-binding EV protein has a signal peptide at its terminus, the signal peptide is cleaved following inserting into the lipid bilayer and incorporation of the IL6/IL6 receptor alpha complex -binding EV protein (now called mature or processed EV protein). Similar to the VEGF-binding EV protein, flexible linkers rich in glycine and serine residues may be present, separating different functional portions of the L6/IL6 receptor alpha- binding EV protein. Alternatively, different functional domains may be directly connected to each other without an intervening linker. An immunoglobulin heavy chain constant region 3 (CH3) may be present between the GP130 protein portion and the chimeric vesicle localization moiety. The CFI3 domain may be linked to the N-terminal GP130 protein portion by glycine-serine rich linker; whereas, the C-terminus of the CH3 domain may be linked to the surface domain of the surface-and- transmembrane domain of the chimeric vesicle localization moiety through a second glycine-serinc rich linker. The chimeric vesicle localization moiety shown in the figure comprises the surface-and- transmembrane domain of Lamp2B and lacks the cytosolic domain of Lamp2B, but instead has the cytosolic domain of PTGFRN. Lastly, an epitope tag may be present in the mature or processed IL6/1L6 receptor alpha-binding EV protein either at the N-terminus or C-terminus. When present in the IL6/IL6 receptor alpha-binding EV protein used in the examples, the epitope tag is present in the N-terminus. Prior to incorporation into an EV or exosome, the N-terminally located epitope tag may be found between the signal peptide and the GP130 protein portion, as in a newly translated or nascent IL6/IL6 receptor alpha-binding EV protein.

[212] All publications, gene transcript identifiers, patents and patent applications discussed and cited herein are incorporated herein by reference in their entireties. It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[213] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[214] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

What is claimed is:

1) A extracellular vesicle for inhibiting IL6 trans-signaling and VEGF-signaling comprising:

(a) an IL6/IL6 receptor alpha complex-binding extracellular vesicle (EV) protein comprising at least domains 1 , 2 and 3 of GP130, for binding IL6/IL6 receptor alpha complex, and a chimeric vesicle localization moiety, and/or

(b) a VEGF-binding EV protein comprising at least two VEGFR-domain fusion units, for binding VEGF, and a chimeric vesicle localization moiety, wherein each chimeric vesicle localization moiety comprises: (i) a surface-and- transmembrane domain of a first vesicle localization moiety, and (ii) a cytosolic domain of a second vesicle localization moiety.

2) The extracellular vesicle of claim 1 , wherein the IL6/IL6 receptor alpha complex-binding EV protein additionally comprises GP130 domain 4, domains 4 and 5, or domains 4, 5 and 6, such that the GP130 domain 1-3 protein portion is extended in the carboxyl direction resulting in a GP130 domain 1-4, domains 1-5, or domains 1-6 protein portion, respectively, of a full length GP130 protein.

3) The extracellular vesicle of claim 1, wherein each VEGFR-domain fusion unit comprises an Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) linked to an Ig-like domain 3 of VEGF receptor 2 (VEGFR-2).

4) The extracellular vesicle of claim 3, wherein the GP130 is a mammalian GP130 and the mammalian GP130 protein is human GP130 protein, its homologue from a mammal, or a variant with at least 85% sequence identity to human GP130 domain 1-3, domain 1-4, domain 1-5, or domain 1-6 protein portion.

5) The extracellular vesicle of claim 4, wherein full length human GP130 comprises an amino acid sequence as provided in NCB1 Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1, or an amino acid sequence of SEQ ID NO: 40 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. 6) The extracellular vesicle of claim 2, wherein the GP130 domain 1-3 protein portion comprises an amino acid sequence starting from glutamic acid at position 23 to aspartic acid at position 324 of NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1 or an amino acid sequence of SEQ ID NO: 41 or a sequence comprising at least 70% sequence identity thereto.

7) The extracellular vesicle of claim 2, wherein the GP130 domain 1-4 protein portion comprises an amino acid sequence starting from glutamic acid at position 23 to histidine at position 425 of NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1 or an amino acid sequence of SEQ ID NO: 42 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

8) The extracellular vesicle of claim 2, wherein the GP130 domain 1-5 protein portion comprises an amino acid sequence starting from glutamic acid at position 23 to alanine at position 517 ofNCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1, or an amino acid sequence of SEQ ID NO: 43 or a sequence comprising at least 70% sequence identity thereto.

9) The extracellular vesicle of claim 2, wherein the GP130 domain 1 -6 protein portion comprises an amino acid sequence starting from glutamic acid at position 23 to glutamic acid at position 619 of NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1 , or an amino acid sequence of

SEQ ID NO: 44 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

10) The extracellular vesicle of claim 4, wherein GP130 domain 1 comprises a conserved glutamine-asparagine dipeptide found within about 150 amino acids from N-terminus of GP130 and/or comprises threonine, arginine, or glutamine eleven amino acids upstream of the conserved glutamine-asparagine dipeptide.

1 1 ) The extracellular vesicle of claim 10, wherein the threonine, arginine, or glutamine may be mutated to a tyrosine; and in which the glutamine-asparagine dipeptide may be mutated singly or doubly, thereby replacing glutamine with phenylalanine and/or replacing asparagine with leucine, so as to increase binding affinity of the IL6/IL6 receptor alpha complex-binding EV protein for IL6/IL6 receptor alpha complex. 12) The extracellular vesicle of claim 10, wherein the GP130 domain 1 comprises an amino acid sequence starting from aspartic acid at position 26 to isoleucine at position 120 of NCBI Reference Sequence: NP_002175.2, or UniProtKB Accession number: P40189-1, or an amino acid sequence of SEQ ID NO: 45 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

13) The extracellular vesicle of claim 11, wherein all three amino acids, one amino acid upstream of the conserved glutamine-asparagine dipeptide as well as the conserved glutamine-asparagine dipeptide, are mutated to a tyrosine, phenylalanine and leucine, respectively, so as to increase binding affinity of the IL6/IL6 receptor alpha complex-binding EV protein for IL6/IL6 receptor alpha complex.

14) The extracellular vesicle of claim 12, wherein the threonine, arginine, or glutamine upstream of the conserved glutamine-asparagine dipeptide is threonine at amino acid position 102 and the glutamine-asparagine dipeptide is at amino acid positions 113 and 1 14 of NCBI Reference Sequence: NP 002175.2, or UniProtKB Accession number: P40189-1 , or threonine at amino acid position 77 and glutamine-asparagine at amino acid positions 88 and 89 of SEQ ID NO: 45 or a sequence comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

15) The extracellular vesicle of claim 14, wherein the threonine- 102 is mutated to tyrosine (T102Y), the glutamine- 113 of the dipeptide is mutated to phenylalanine (QI 13F), or the asparagine- 114 of the dipeptide is mutated to leucine (N114L), or a combination thereof, to increase binding affinity of the IL6/IL6 receptor alpha complex-binding EV protein for IL6/IL6 receptor alpha complex.

16) The extracellular vesicle of claim 1, wherein the chimeric vesicle localization moiety of the 1L6/1L6 receptor alpha complex-binding EV protein comprises the surface-and- transmembrane domain of LAMP2b and the cytosolic domain of PTGFRN.

17) The extracellular vesicle of claim 1, wherein the chimeric vesicle localization moiety comprises a first and second vesicle localization moieties of the IL6/IL6 receptor alpha complex-binding EV protein which are human.

18) The extracellular vesicle of claim 16, wherein the surface-and-transmembrane domain of

LAMP2b of the IL6/IL6 receptor alpha complex-binding EV protein comprises an amino acid sequence starting from leucine at position 29 to alanine at position 395 of NCBI Reference Sequence Accession number NP 054701 .1 , or an amino acid sequence of SEQ ID NO: 46 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

19) The extracellular vesicle of claim 16, wherein the cytosolic domain of PTGFRN of the IL6/IL6 receptor alpha complex-binding EV protein comprises an amino acid sequence starting from serine at position 854 to aspartic acid at position 879 of NCBI Reference Sequence Accession number NP 065173.2, or an amino acid sequence of SEQ ID NO: 47 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

20) The extracellular vesicle of claim 1, wherein the chimeric vesicle localization moiety of the IL6/IL6 receptor alpha complex-binding EV protein comprises the surface-and- transmembrane domain of LAMP2b and the cytosolic domain of PTGFRN comprising an amino acid sequence of SEQ ID NO: 48 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

21) The extracellular vesicle of claim 1, wherein the IL6/IL6 receptor alpha complex-binding EV protein additionally comprises a third constant immunoglobulin domain, CH3 domain, of IgGl heavy chain.

22) The extracellular vesicle of claim 21 , wherein the IgGl heavy chain is human.

23) The extracellular vesicle of claim 21, wherein the CH3 domain of human IgGl heavy chain comprises an amino acid sequence starting from glycine at position 110 to lysine at position 216 of GenBank Accession Number: AAL96263.1 , or an amino acid sequence of SEQ ID NO: 49 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

24) The extracellular vesicle of claim 1, wherein the IL6/IL6 receptor alpha complex-binding EV protein additionally comprises a flexible linker.

25) The extracellular vesicle of claim 24, wherein the flexible linker is a glycine-serine rich linker. 26) The extracellular vesicle of claim 25, wherein the glycine-serine rich linker is selected from the group consisting of (GS)₃ (SEQ ID NO: 50), (GS)₄ (SEQ ID NO: 51), (GS)₅ (SEQ ID NO: 52), (GS)₆ (SEQ ID NO: 53), (GS)₇ (SEQ ID NO: 54), (GS)₈ (SEQ ID NO: 55), (GS)₉ (SEQ ID NO: 56), (GS)io (SEQ ID NO: 57), (GGS)₂ (SEQ ID NO: 58), (GGS)₃ (SEQ ID NO: 59), (GGS)₄ (SEQ ID NO: 60), (GGS)₅ (SEQ ID NO: 61), (GGS)₆ (SEQ ID NO: 62), (GGGS)₂ (SEQ ID NO: 63), (GGGS)₃ (SEQ ID NO: 64), (GGGS)₄ (SEQ ID NO: 65), (GGGS)s (SEQ ID NO: 66), (GGGGS)₂ (SEQ ID NO: 67), (GGGGS)₃ (SEQ ID NO: 68), (GGGGS)₄ (SEQ ID NO: 69), (GGGGGS) (SEQ ID NO: 70), (GGGGGS)₂ (SEQ ID NO: 71), (GGGGGS)₃ (SEQ ID NO: 72), (SG)₂S (SEQ ID NO: 73), (SG)₃S (SEQ ID NO: 74), (SG)₄S (SEQ ID NO: 75), (SG)₅S (SEQ ID NO: 76), (SG)₆S (SEQ ID NO: 77), (SG)₇S (SEQ ID NO: 78), (SG)gS (SEQ ID NO: 79), and (SG)₉S (SEQ ID NO: 80), or a portion thereof.

27) The extracellular vesicle of claim 25, wherein the glycine-serine rich linker is any of (GS)₃ (SEQ ID NO: 50), (GS)₅ (SEQ ID NO: 52), (SG)₄S (SEQ ID NO: 75), and (GGGGS)₃ (SEQ ID NO: 68).

28) The extracellular vesicle of claim 1, wherein a GP130 protein portion and the surface domain of the chimeric vesicle localization moiety are found on the outer surface or external to the extracellular vesicle , the transmembrane domain of the chimeric vesicle localization moiety is found in the lipid bilayer of the extracellular vesicle , and the cytosolic domain of the chimeric vesicle localization moiety is found in the inner or interior portion of the extracellular vesicle .

29) The extracellular vesicle of claim 1, wherein the GP130 domain 1-3 protein portion with or without additional GP130 domain 4, 4-5, or 4-6 is linked directly through its C -terminus to the N -terminus of the chimeric vesicle localization moiety.

30) The extracellular vesicle of claim 1, wherein the IL6/IL6 receptor alpha complex-binding EV protein is free of GP130 transmembrane domain.

31) The extracellular vesicle of claim 1, wherein the IL6/IL6 receptor alpha complex-binding EV protein is free of GP130 cytoplasmic domain.

32) The extracellular vesicle of claim 1, wherein the IL6/IL6 receptor alpha complex-binding EV protein is free of both GP130 transmembrane domain and cytoplasmic domain. 33) The extracellular vesicle of claim 1, wherein the IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-human GP130 portion-human Lamp2b extracellular domain-and-transmcmbrane domain-cytosolic domain of human PTGFRN-C terminus, in an extracellular vesicle , wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex.

34) The extracellular vesicle of claim 1 or 33, the IL6/IL6 receptor alpha complex-binding EV protein comprises an amino acid sequence of mature MB20 from amino acid at position 22 to end of SEQ ID NO: 20 or mature MB22 from amino acid at position 22 to end of SEQ ID NO: 22.

35) The extracellular vesicle of claim 27, wherein the GP130 domain 1-3 protein portion with or without additional GP130 domain 4, 4-5, or 4-6 is linked through a glycine-serine rich linker to the N-terminus of the chimeric vesicle localization moiety.

36) The extracellular vesicle of claim 35, wherein the glycine-serine rich linker is (GGGGS)3 (SEQ ID NO: 68).

37) The extracellular vesicle of claim 1, wherein the 11,6/11,6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-human GP130 portion-linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, in an extracellular vesicle or exosome, wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex.

38) The extracellular vesicle or exosome of claim 1 or 37, the IL6/IL6 receptor alpha complex- binding EV protein comprises an amino acid sequence of a mature MB 16 from amino acid at position 22 to end of SEQ ID NO: 16 or a mature MB 18 from amino acid at position 22 to end of SEQ ID NO: 18.

39) The extracellular vesicle of claim 1 , wherein the GP130 domain 1-3 protein portion with or without additional GP130 domain 4, 4-5, or 4-6 is linked to the N-terminus of the chimeric vesicle localization moiety through glycine-rich linkers attached to both ends of the CH3 domain of human IgGl heavy chain. 40) The extracellular vesicle of claim 39, wherein the glycine-rich linkers attached to both ends of the CH3 domain of human IgGl heavy chain comprise the (GS)3 linker (SEQ ID NO: 50) attached to the N-terminus of the CH3 domain and the (GS)5 linker (SEQ ID NO: 52) attached to the C -terminus of the CH3 domain.

41) The extracellular vesicle of claim 1, wherein the IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-human GP130 portion-linker-CH3 domain of human IgGl -linker-human Lamp2b extracellular domain-and- transmembrane domain-cytosolic domain of human PTGFRN-C terminus, into an extracellular vesicle , wherein the GP130 domain 1 portion optionally may have one or more mutations which enhance binding to IL6/IL6R complex.

42) The extracellular vesicle of claim 1 , wherein the IL6/IL6 receptor alpha complex-binding EV protein comprises an amino acid sequence of a mature MB02 from amino acid at position 22 to end of SEQ ID NO: 2, a mature MB04 from amino acid at position 22 to end of SEQ ID NO: 4, a mature MB06 from amino acid at position 22 to end of SEQ ID NO: 6, or a mature MB08 from amino acid at position 22 to end of SEQ ID NO: 8.

43) The extracellular vesicle of claim 1, wherein the IL6/IL6 receptor alpha complex-binding EV protein from amino-to-carboxyl direction is organized as: N terminus-signal peptide- human GP130 domain 1-5 portion-(GS)a linker-CH3 domain of human IgGl-(GS)s linker- human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, wherein the signal peptide initially present in the nascent or newly synthesized protein is cleaved off in the mature protein and wherein the GP130 domain 1 portion comprises T102Y, QI 13F and N114L to increase binding affinity for IL6/IL6 receptor alpha complex.

44) The extracellular vesicle of claim 43, wherein the 1L6/1L6 receptor alpha complex-binding

EV protein comprises an amino acid sequence of a mature MB02 from amino acid at position 22 to end of SEQ ID NO: 2.

45) The extracellular vesicle of claim 1 , wherein the VEGF-binding EV protein comprising the VEGFR-domain fusion unit comprises an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereof. 46) The extracellular vesicle of claim 45, wherein the amino acid sequence having over 98% sequence identity to the VEGFR-domain fusion unit is selected from the group consisting of sequences homologous to:

(a) the Ig-like domain 2 of a VEGF receptor 1 (VEGFR-1) of SEQ ID NO: 86 present in NCBI Reference Sequence Accession Number or GenBank Accession Number of

XP_055100552.1, XP 034792394.1, XP_008969852.1, XP 032006460.1 , XP 032006459.1 , XP 055100550.1 , XP 003818375.1, PNJ48512.1, XP_032006458.1, PNJ48513.1, XP 055100549.1 , AYL88758.1 , XP 025220259.1, XP 009189959.1 , XP 008020010.1 , XP_015294597.1, XP_028693312.1, XP_025220258.1, XP 015294596.1, XP 030674446.1, XP-003270267.1 , EHH58499.1 , XP 025220257.1 , XP 011940270.1 , XP _003913768.1 , XP 005585612.1, XP_008020007.1, XP_001117928.3, EHH28920.1, XP_003270266.1, XP_033092342.1, XP_023064515.1, XP_011753897.1 , XP 017703974.1, XP 021531864.1, XP_015294597.2, XP 033092333.1, XP 033092324.1, XP 023064514.1, XP_015294596.2, XP 017703973.1, XP 032150285.1, XP 011753896.1, XP 023064513.1, XP 033092313.1 , XP 005585612.2, XP_017703972.1, XP 021531863.1, XP_032150284.1 , XP 010360667.2, XP_010360657.2, XP_030778324.1, XP 017392985.1 , XP_010360648.2, XP_011814493.1 , or XP 017392983.1, and

(b) the Ig-like domain 3 of a VEGF receptor 2 (VEGFR-2) of SEQ ID NO: 87 present in NCBI Reference Sequence Accession Number or GenBank Accession Number of BAD93138.1 , AAI31823.1 , XP_011708980.1 , XP_007996878.1 , XP_005555327.1 , XP_014994176.1, EHH26004.1, XP_031520318.1, EHH53800.1, XP_011928915.1, XP_011847889.1, XP 011708979.1, XP 005555327.2, XP_011928917.1 , XP_025240971.1, XP_003268434.2, XP_032130119.1, XP_012296575.1, XP_002745866.2, XP_032130121.1 , XP 012296576.1, XP_032130122.1, XP_054109329.1, XP 008991591.1, XP 032130120.1 , XP_055150622.1, XP_032007207.1, XP_007121194.1, XP 017395743. 1, XP_028347631.1 , XP_028347630.1 , XP 028347633.1, XP 003933665.1 , XP_017395745.1, XP_028347632.1 , XP 017395746.1 , XP_017395744.1 , XP_010361178.1 , XP_011817095.1 , XP_023048010.1, XP_ 036709011.1, XP 019783716.1, XP 030736423.1, XP 022419698.1, XP 029090747.1, XP 004268311.1, XP_030615937.1, KAJ8795398.1, XP_036709012.1, XP 007168490.2, TKC45787.1, XP 033281298.1, XP_022419718.1, XP_033281299.1, XPJJ22419727.1, XP 030736425.1, XP_030736424.1, XP_019783717.1, XP_019783718.1, XP_042792296.1, XP 033281300.1, XP 019325208.1, XP_047712387.1, XP_049486434.1, XP_042792297.1, XPJM3428557.1, XP_042840444.1, XP_040339255.1, XP_026912793.1, XP_011280181.3, VFV35517.1, XP_030169713.1, XP_046947373.1, XP_007075942.2, XP 047712388.1 , XP 049486433.1, XP_040339249.1, XP_033070538.1 , XP_025777172.1, KAB1282375.1 , XP 006177399.1, and XP_031290309.1.

47) The extracellular vesicle of claim 1, wherein the VEGF-binding EV protein additionally comprises a third constant immunoglobulin domain, CH3 domain, of IgGl heavy chain.

48) The extracellular vesicle of claim 47, wherein the IgGl heavy chain is human.

49) The extracellular vesicle of claim 47, wherein the CH3 domain of human IgGl heavy chain comprises an amino acid sequence starting from glycine at position 1 10 to lysine at position 216 of GenBank Accession Number: AAL96263.1, or an amino acid sequence of SEQ ID NO: 49 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

50) The extracellular vesicle of claim 1, wherein the VEGF-binding EV protein additionally comprises a linker.

51) The extracellular vesicle of claim 50, wherein the linker is a glycine-serine rich linker.

52) The extracellular vesicle of claim 51, wherein the glycine-serine rich linker is selected from the group consisting of (GS)₃ (SEQ ID NO: 50), (GS)₄ (SEQ ID NO: 51), (GS)s (SEQ ID NO: 52), (GS)₆ (SEQ ID NO: 53), (GS)₇ (SEQ ID NO: 54), (GS)₈ (SEQ ID NO: 55), (GS)₉ (SEQ ID NO: 56), (GS)io (SEQ ID NO: 57), (GGS)₂ (SEQ ID NO: 58), (GGS)₃ (SEQ ID NO: 59), (GGS)₄ (SEQ ID NO: 60), (GGS)₅ (SEQ ID NO: 61), (GGS)₆ (SEQ ID NO: 62), (GGGS)₂ (SEQ ID NO: 63), (GGGS)₃ (SEQ ID NO: 64), (GGGS)₄ (SEQ ID NO: 65), (GGGS)s (SEQ ID NO: 66), (GGGGS)₂ (SEQ ID NO: 67), (GGGGS)₃ (SEQ ID NO: 68), (GGGGS)₄ (SEQ ID NO: 69), (GGGGGS) (SEQ ID NO: 70), (GGGGGS)₂ (SEQ ID NO: 71), (GGGGGS)₃ (SEQ ID NO: 72), (SG)₂S (SEQ ID NO: 73), (SG)₃S (SEQ ID NO: 74), (SG)₄S (SEQ ID NO: 75), (SG)₅S (SEQ ID NO: 76), (SG)₆S (SEQ ID NO: 77), (SG)₇S (SEQ ID NO: 78), (SG)₈S (SEQ ID NO: 79), and (SG)₉S (SEQ ID NO: 80), or a portion thereof. 53) The extracellular vesicle of claim 51 , wherein the glycine-serine rich linker is any of (GS)₃ (SEQ ID NO: 50), (GS)₅ (SEQ ID NO: 52), (SG)₄S (SEQ ID NO: 75), and (GGGGS)₃ (SEQ ID NO: 68).

54) The extracellular vesicle of claim 49, wherein the flexible linker is between VEGFR-domain fusion units in a head-to-tail concatemer and between an adjacent VEGFR-domain fusion unit and the chimeric vesicle localization moiety.

55) The extracellular vesicle of claim 54, wherein the VEGFR-domain fusion units in a head-to- tail concatemer comprises an arrangement from N-to-C-terminus: [[(VEGFR-1 domain 2)- (VEGFR-2 domain 3 )]-linker]_n- [(VEGFR-1 domain 2)-(VEGFR-2 domain 3)], where n = 1 for a concatemer comprising 2 VEGFR-domain fusion units, n = 2 for a concatemer comprising 3 VEGFR-domain fusion units, and n = integer i for a concatemer comprising i + 1 VEGFR-domain fusion units.

56) The extracellular vesicle of claim 55, wherein the linker is selected from a group consisting of (SG)₄S, (GS)₅, and (GGGGS)₃.

57)The extracellular vesicle of claim 55, wherein the linker is (SG)₄S.

58) 1'he extracellular vesicle of claim 1, wherein the VEGF-binding EV protein additionally comprises a signal peptide at its N-terminus for insertion and passage of the VEGFR-domain fusion units and the surface domain of the chimeric vesicle localization moiety through a lipid bilayer of an extracellular vesicle or an exosome.

59) The extracellular vesicle of claim 1, wherein a VEGFR-domain fusion units and the surface domain of the chimeric vesicle localization moiety arc found on the outer surface or external to the extracellular vesicle , the transmembrane domain of the chimeric vesicle localization moiety is found in the lipid bilayer of the extracellular vesicle , and the cytosolic domain of the chimeric vesicle localization moiety is found in the inner or interior portion of the extracellular vesicle .

60) The extracellular vesicle of claim 1, wherein the chimeric vesicle localization moiety of the VEGF-binding EV protein comprises the surface-and-transmembrane domain of LAMP2b and the cytosolic domain of PTGFRN. 61) The extracellular vesicle of claim 1, wherein the chimeric vesicle localization moiety comprises a first and second vesicle localization moieties of the VEGF-binding EV protein which are human.

62) The extracellular vesicle of claim 60, wherein the surface-and-transmembrane domain of LAMP2b of the VEGF-binding EV protein comprises an amino acid sequence starting from leucine at position 29 to alanine at position 395 of NCBI Reference Sequence Accession number NP_054701.1, or an amino acid sequence of SEQ ID NO: 46 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

63) The extracellular vesicle of claim 60, wherein the cytosolic domain of PTGFRN of the VEGF-binding EV protein comprises an amino acid sequence starting from serine at position 854 to aspartic acid at position 879 of NCBI Reference Sequence Accession number NP 065173.2, or an amino acid sequence of: SEQ ID NO: 47 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

64) The extracellular vesicle of claim 1 , wherein the chimeric vesicle localization moiety of the VEGF-binding EV protein comprises the surface-and-transmembrane domain of LAMP2b and the cytosolic domain of PTGFRN comprising an amino acid sequence of SEQ ID NO: 48 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

65) The extracellular vesicle of claim 1, wherein the two or more VEGFR-domain fusion units or the VEGFR-domain fusion unit concatemer are linked to the chimeric vesicle localization moiety through a flexible linker.

66) The extracellular vesicle of claim 65, wherein the linker is a glycine-serine rich linker.

67) The extracellular vesicle of claim 66, wherein the glycine-serine rich linker is (GGGGS)3.

68) The extracellular vesicle of claim 1, wherein the VEGF-binding EV protein from amino-to- carboxyl direction is organized as: N terminus -VEGFR-domain fusion units-linker-human Lamp2b extracellular domain-and-transmembrane domain-cytosolic domain of human PTGFRN-C terminus, (mature VEGF-binding EV protein) into an extracellular vesicle . 69) The extracellular vesicle of claim 1, wherein the VEGF-binding EV protein comprises an amino acid sequence of a mature MB 14 from amino acid number 22 to end of SEQ ID NO: 14 or a mature MB25 from amino acid 22 to end of SEQ ID NO: 26.

70) The extracellular vesicle of claim 1, wherein the two or more VEGFR-domain fusion units or the VEGFR-domain fusion unit concatemer are linked to the chimeric vesicle localization moiety through a peptide comprising from N-to-C direction a 1^st flexible linker, the CH3 domain, and a 2^nd flexible linker.

71) The extracellular vesicle of claim 70, wherein the first flexible linker and second flexible linker are glycine-serine rich linkers.

72) The extracellular vesicle of claim 71 , wherein the first flexible linker is (SG)4S (SEQ ID

NO: 75).

73) The extracellular vesicle of claim 71, wherein the second flexible linker is (GS)s (SEQ ID NO: 52).

74) The extracellular vesicle of claim 1, wherein the VEGF-binding EV protein from amino-to- carboxyl direction is organized as: N terminus-VEGFR-domain fusion units-first linker-CH3 domain-second linker-human Lamp2b extracellular domain-and-transmembrane domain- cytosolic domain of human PTGFRN-C terminus (mature VEGF-binding EV protein) into an extracellular vesicle .

75) The extracellular vesicle of claim 1, wherein the VEGF-binding EV protein comprises an amino acid sequence of a mature MB 10 from amino acid position 22 to the end of SEQ ID NO: 10 or a mature MB 12 from amino acid position 22 to the end of SEQ ID NO: 12 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

76) The extracellular vesicle of claim 1, comprising: (a) the IL6/IL6 receptor alpha complex- binding EV protein is selected from the group consisting of a mature MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, a mature MB06 comprising an amino acid sequence from position 22 to end of SEQ ID NO:6, a mature MB08 comprising an amino acid sequence from position 22 to end of SEQ ID NO:8, a mature MB 16 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 16, a mature MB 18 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 18, a mature MB20 comprising an amino acid sequence from position 22 to end of SEQ ID NO:20, and a mature MB22 comprising an amino acid sequence from position 22 to end of SEQ ID NO:22, and (b) the VEGF-binding EV protein is selected from the group consisting of a mature MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10, a mature MB 12 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 12, a mature MB12 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 14, and a mature MB25 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 26 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

77) The extracellular vesicle of claim 1, comprising: (a) the IL6/IL6 receptor alpha complex- binding EV protein selected from the group consisting of a mature MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, a mature MB08 comprising an amino acid sequence from position 22 to end of SEQ ID NO:8, a mature MB 16 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 16, and a mature MB20 comprising an amino acid sequence from position 22 to end of SEQ ID NO:20, and (b) the VEGF-binding EV protein selected from the group consisting of a mature MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10, a mature MB 12 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 12, a mature MB14 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 14, and a mature MB25 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 26 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

78) The extracellular vesicle of claim 1, comprising: (a) the IL6/IL6 receptor alpha complex- binding EV protein, a mature MB 02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, and (b) the VEGF-binding EV protein selected from the group consisting of a mature MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10, a mature MB 12 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 12, a mature MBH comprising an amino acid sequence from position 22 to end of SEQ ID NO: 14, and a mature MB25 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 26 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

79) The extracellular vesicle of claim 1 , comprising: (a) the IL6/IL6 receptor alpha complex- binding EV protein selected from the group consisting of a mature MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, a mature MB08 comprising an amino acid sequence from position 22 to end of SEQ ID NO:8, a mature MB 16 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 16, and a mature MB20 comprising an amino acid sequence from position 22 to end of SEQ ID NO:20, and (b) the VEGF-binding EV protein, a mature MB25 (SEQ ID NO: 39).

80) The extracellular vesicle of claim 1, comprising: (a) the IL6/IL6 receptor alpha complex- binding EV protein selected from the group consisting of a mature MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, a mature MB08 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 8, a mature MB 16 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 16, and a mature MB20 comprising an amino acid sequence from position 22 to end of SEQ ID NO:20, and (b) the VEGF-binding EV protein, a mature MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10.

81) The extracellular vesicle of claim 1 , comprising: (a) the IL6/IL6 receptor alpha complex- binding EV protein, a mature MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, and (b) the VEGF-binding EV protein, a mature MB25 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 26.

82) The extracellular vesicle of claim 1, comprising: (a) the IL6/IL6 receptor alpha complex- binding EV protein, a mature MB02 comprising an amino acid sequence from position 22 to end of SEQ ID NO:2, and (b) the VEGF-binding EV protein, a mature MB 10 comprising an amino acid sequence from position 22 to end of SEQ ID NO: 10.

83) A composition comprising a plurality of the extracellular vesicles of claim 1 and a suitable carrier.

84) The composition of claim 83, wherein about 75% of the extracellular vesicles are positive for both the mature MB02 and the mature MB010 proteins. 85) The composition of claim 83, wherein mode of IL6/IL6 receptor alpha complex-binding EV protein molecules per extracellular vesicle positive for the IL6/IL6 receptor alpha complex- binding EV protein is at about 120 molecules.

86) The composition of claim 83, wherein the extracellular vesicles inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 8.22E+08 EV/mL to 3.22E+10 EV/mL.

87) The composition of claim 83, wherein the extracellular vesicles inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 1 .73E+10 EV/mL.

88) The composition of claim 83, wherein the extracellular vesicles inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 8.22E+08 EV/mL.

89) The composition of claim 83, wherein the VEGF-binding EV protein binds VEGF inhibiting VEGF-signaling or angiogenesis and wherein the extracellular vesicles inhibit VEGF signaling in HUVEC cells with an IC50 of about 1.95E+09 EV/mL to 6.88E+09 EV/mL.

90) The composition of claim 83, wherein the extracellular vesicles inhibit VEGF signaling in HUVEC cells with an IC50 of about 1.95E+09 EV/mL to 2.81E+09 EV/mL.

91) The composition of claim 83, wherein the extracellular vesicles inhibit VEGF signaling in

HUVEC cells with an IC50 of about 2.81E+09 EV/mL.

92) The composition of claim 83, wherein the extracellular vesicle inhibits VEGF signaling in HUVEC cells with an IC50 of about 1.95E+09 EV/mL.

93) The composition of claim 83, wherein the extracellular vesicles simultaneously bind both IL6-IL6 receptor complex inhibiting IL6-trans-signaling and binds VEGF inhibiting VEGF- signaling.

94) The composition of claim 83, wherein the extracellular vesicles bind multiple IL6-IL6 receptor complexes and multiple copies of VEGF.

95) The composition of claim 83, wherein the extracellular vesicles inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 3.22E+10 EV/mL and VEGF signaling in HUVEC cells with an IC50 of about 2.81E+09 EV/mL. 96) The composition of claim 83, wherein the extracellular vesicles inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 3.22E+10 EV/mL and VEGF signaling in HUVEC cells with an IC50 of about 1.95E+09 EV/mL.

97) The composition of claim 83, wherein the extracellular vesicles inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 8.22E+08 EV/mL and VEGF signaling in HUVEC cells with an IC50 of about 2.81E+09 EV/mL.

98) The composition of claim 83, wherein the extracellular vesicles inhibit IL6-trans-signaling in HUVEC cells with an IC50 of about 8.22E+08 EV/mL and VEGF signaling in HUVEC cells with an IC50 of about 1.95E+09 EV/mL.

99) The composition of claim 83, wherein the VEGF-binding EV protein comprises 3 VEGFR- domain fusion units, so as to maintain VEGF function in the presence of the IL6/IL6 receptor alpha complex-binding EV protein.

100) A method for obtaining the extracellular vesicle of claim 1 for inhibiting IL6 trans- signaling and inhibiting VEGF-signaling, comprising culturing an extracellular vesicle - producing cell comprising the vector for expressing the IL6/IL6 receptor alpha complex- binding EV protein and VEGF-binding EV protein and harvesting culture medium of the extracellular vesicle -producing cell, thereby obtaining the extracellular vesicle of claim 1.

101) A kit comprising the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF- binding EV protein of claim 1 or composition of claim 83 and a label.

102) A method for inhibiting both IL6 trans-signaling and VEGF signaling in a subject comprising administering an effective amount of a extracellular vesicle of claim 1 or a composition of claim 83, so as to inhibit both IL6 trans-signaling and VEGF signaling in the subject, thereby inhibiting both IL6 trans-signaling and VEGF signaling in the subject.

103) A method for reducing both 1L6 trans-signaling and VEGF signaling in a subject comprising administering an effective amount of a extracellular vesicle of claim 1 or a composition of claim 83, so as to reduce both IL6 trans-signaling and VEGF signaling in the subject, thereby reducing both IL6 trans-signaling and VEGF signaling in the subject.

104) A method for treating inflammation associated with IL6 trans-signaling and angiogenesis associated with VEGF signaling in a subject comprising administering an effective amount of a extracellular vesicle of claim 1 or a composition of claim 83, so as to simultaneously inhibit or reduce IL6 trans-signaling and VEGF signaling in the subject, treating inflammation associated with IL6 trans-signaling and angiogenesis associated with VEGF signaling in the subject

105) The method of claim 104, wherein the subject is a subject afflicted with a tumor, a cancer, a first condition of acute or chronic inflammation due to overactive IL6 trans-signaling, a second condition of angiogenesis or neovascularization from overactive VEGF signaling or excess VEGF level, or a combination thereof.

106) The method of claim 105, wherein the first and/or second condition is any of diabetic retinopathy, retinal vein occlusion, uveitis, choroidal neovascularization, diabetic macular edema, dry eye, dry age-related macular degeneration (AMD), wet AMD, and retinopathy of prematurity.

107) The method of claim 102, 103, or 104, wherein the IL6/IE6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein are mature proteins lacking a signal peptide.

108) The method of claim 102, 103, or 104, wherein the IL6/IL6 receptor alpha complex-binding EV protein and the VEGF-binding EV protein are formulated as a composition of claim 83.

109) A method for inhibiting an eye disease by providing a reservoir of an active agent for treating, inhibiting, or reducing inflammation and angiogenesis in an eye of a subject in need thereof comprising intravitreal injection of the extracellular vesicle of claim 1 or a composition of claim 83 into the eye of the subject, so that the extracellular vesicles reside in the vitreous humor and retina, thereby providing the reservoir of the active agent for treating, inhibiting, or reducing inflammation and angiogenesis in the eye of the subject.

110) The method of claim 109, wherein the subject is a subject afflicted with a tumor, a cancer, a condition of acute or chronic inflammation due to overactive IL6 trans signaling, a condition of neo-angiogenesis from overactive VEGF signaling or excess VEGF level, or a combination thereof. I l l) The method of claim 110, wherein the disease is any of diabetic retinopathy, retinal vein occlusion, uveitis, choroidal neovascularization, diabetic macular edema, dry eye, dry age- related macular degeneration (AMD), wet AMD, and retinopathy of prematurity.

112) A method for treating, inhibiting, or reducing uveitis in an eye of a subject comprising administering an intravitreal injection of the extracellular vesicle of claim 1 or the composition of claim 83 in an effective amount so as to treat, inhibit, or reduce uveitis in the eye of the subject, thereby treating, inhibiting, or reducing uveitis in the eye of the subject.

1 13) A method for treating, inhibiting, or reducing choroidal neovascularization (CNV) in an eye of a subject comprising administering an intravitreal injection of the extracellular vesicle of claim 1 or the composition of claim 83 in an effective amount so as to treat, inhibit, or reduce CNV in the eye of the subject, thereby treating, inhibiting, or reducing CNV in the eye of the subject.

114) The method of claim 113, wherein the CNV is located in the posterior of the eye.

115) The method of claim 114, wherein the CNV is near or around an optic nerve.

1 16) The method of claim 1 13, wherein the treating, inhibiting, or reducing CNV comprises reduction in vascular leakage.

117) The method of claim 113, wherein CNV is associated with wet AMD.

118) The method of claim 102, 103, 104, 109, 112, or 113, wherein the subject is a mammal.

119) The method of claim 118, wherein the mammal is a human.