Genetically reprogrammed exosomes for immunotherapy of acute myeloid leukemia

Abstract

Engineered extracellular vesicle having a first fusion protein comprising an anti-CD3 antibody moiety, an anti-CLL-1 antibody moiety, and a first transmembrane domain, a second 5 fusion protein comprising a PD-1 protein, a second transmembrane domain, and a CD70 protein, such that both the first fusion protein and the second fusion protein are displayed on a surface of the engineered extracellular vesicle, and methods of using the same.

Description

[0001] GENETICALLY REPROGRAMMED EXOSOMES FOR IMMUNOTHERAPY OF ACUTE MYELOID LEUKEMIA

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U. S. C. § 119(e) of U. S. Provisional Application No.: 63 / 729,681, filed December 9, 2024, the contents of which are incorporated by reference.

[0004] STATEMENT REGARDING FEDERAL FUNDING

[0005] This invention was made with government support under W81XWH-19-1-0272 awarded by the Defense Health Agency, Medical Research and Development Branch, and CA276240, and EB031830 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Acute myeloid leukemia (AML) is a form of bone marrow and blood cancer that is characterized by an increased number of undifferentiated myeloblasts. AML occurs when leukemia affects the myeloid cells in the bone marrow which under normal conditions, turn into red blood cells, white blood cells, and platelets. As a result, leukemia cells proliferate rapidly in the bone marrow and blood and migrate to other parts of the body, including the central nervous system (brain and spinal cord) and skin. AML progresses quickly and may eventually lead to fatal complications of infection, bleeding, or organ infiltration, often within weeks. AML occurs in both adults and children, and the five-year survival rate is less than 25% for adults. AML accounts for over 13,000 new cases of leukemia each year.

[0008] Accordingly, there is an urgent need to develop new therapeutic approaches for leukemias, and in particular, AML. The present invention satisfies this need.

[0009] SUMMARY OF THE INVENTION

[0010] Current treatments for acute myeloid leukemia (AML) remain challenging, characterized by poor clinical outcomes. Exosomes, cell-derived membranous vesicles, have been emerging as new modalities of therapy. In the present disclosure, genetically reprogrammed exosomes were designed and generated with surface displayed antibodies and immunoregulatory proteins, namely programmed immune-engaging exosomes (PRIME Exos). By simultaneously targeting T cells and AML cells expressing C-type lectin-like molecule- 1 (CLL-1), PRIME Exos can elicit tumor-specific immune responses and sustain cellular

[0011] 1

[0012] 530.045W01

[0013] 2024-123-02 immunity against AML through modulating programmed death 1 (PD-1)- and CD27-mediated immune checkpoint pathways. In preclinical models of AML, PRIME Exos demonstrate promising efficacy and safety for suppressing leukemia expansion. This study developed a new exosome-based approach for AML immunotherapy.

[0014] Accordingly, in some embodiments, the disclosure provides for engineered extracellular vesicle comprising a first fusion protein comprising a formula A-B-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second fusion protein comprising the formula D-E-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; and wherein both the first fusion protein and the second fusion protein are displayed on a surface of the engineered extracellular vesicle.

[0015] In some embodiments, the disclosure provides for engineered extracellular vesicle comprising a first fusion protein comprising a formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second fusion protein comprising the formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; wherein both the first fusion protein and the second fusion protein are displayed on a surface of the engineered extracellular vesicle; and wherein L1 – L4 are optional linker sequences.

[0016] In some embodiments, each of the first antibody moiety and the second antibody moiety comprise a single chain variable fragment (scFv), a single domain antibody, a bispecific antibody, or a multi-specific antibody. In some embodiments, each of the first antibody moiety and the second antibody moiety comprise a single chain variable fragment (scFv).

[0017] In some embodiments, the first transmembrane domain comprises a transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR) protein and / or the second transmembrane domain comprises a transmembrane domain of a CD9 protein. In some embodiments, the first transmembrane domain comprises a transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR) protein and the second transmembrane domain comprises a transmembrane domain of a CD9 protein.

[0018] In some embodiments, the first fusion protein and the second fusion protein are displayed on the surface of the engineered extracellular vesicle in a 1:1 ratio.

[0019] In some embodiments, the extracellular vesicle comprises one or more of an exosome, a liposome, a microvesicle, an apoptotic body, and a combination thereof. In some embodiments, the engineered extracellular vesicle comprises a particle size of about 5 nm to

[0020] 2

[0021] 530.045W01

[0022] 2024-123-02 about 500 nm, about 10 nm to about 400 nm, about 10 nm to about 300 nm, about 10 nm to about 250 nm, or about 10 nm to about 150 nm.

[0023] In some embodiments, formula A-L1-B-L2-C can be formula A-B-C wherein A is an N-terminus and C is the C-terminus of the first fusion protein. In some embodiments, formula D-L1-E-L2-F can be formula A-B-C wherein D is an N-terminus and F is the C-terminus of the second fusion protein.

[0024] In some embodiments, the first fusion protein and the second fusion protein comprise one or more of the linker sequences Li - L4, wherein the linker sequences Li - L4 comprise the amino acid sequence (GGGS)n (SEQ ID NO: 48), where n is an integer between 1 and 5. In other embodiments, first fusion protein and the second fusion protein comprise the linker sequences Li - L4, wherein the linker sequences Li - L4 comprise amino acid sequence (GGGS)n (SEQ ID NO: 48), where n is an integer from 1 to 5. Preferably, at least one of the linker sequences Li - L4 comprises (GGGS)2 (SEQ ID NO: 49).

[0025] In some embodiments, the first fusion protein and the second fusion protein comprise one or more of the linker sequences Li - L4, wherein the linker sequences L1 – L4 comprise the amino acid sequence (GGGGS)n (SEQ ID NO: 50), where n is an integer between 1 and 5. In other embodiments, first fusion protein and the second fusion protein comprise the linker sequences Li - L4, wherein the linker sequences Li - L4 comprise amino acid sequence (GGGGS)n (SEQ ID NO: 50), where n is an integer from 1 to 5. Preferably, at least one of the linker sequences Li - L4 comprises (GGGGS)₂ (SEQ ID NO: 53).

[0026] In some embodiments, the first fusion protein and the second fusion protein further comprise a hemagglutinin epitope tag and / or a histidine epitope tag.

[0027] In some embodiments, the first fusion protein comprises an amino acid sequence that is at least about 60% identical to about 100% identical or about 85% to about 100% identical to SEQ ID NO. 1 or 13, or about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 99% identical or identical to SEQ ID NO: 1 or 13; and the second fusion protein comprises an amino acid sequence that is at least about 60% identical to about 100% identical or about 85% to about 100% identical to SEQ ID NO. 2 or 14, or about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 99% identical or identical to SEQ ID NO: 2 or 14.

[0028] 3

[0029] 530.045W01

[0030] 2024-123-02 In other embodiments, the first fusion protein comprises an amino acid sequence identical to SEQ ID NO: 1 or 13; and the second fusion protein comprises an amino acid sequence identical to SEQ ID NO: 2 or 14.

[0031] The disclosure also provides for composition comprising an engineered extracellular vesicle as described herein; and a pharmaceutically acceptable carrier. For example, a composition may comprise a first engineered exosome and a second engineered exosome, the first engineered exosome comprising a first fusion protein comprising formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; the second engineered exosome comprising a second fusion protein comprising the formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; wherein both the first fusion protein and the second fusion protein are displayed on a surface of the first engineered exosome and the second engineered exosome, respectively; and wherein Li - L4 are optional linker sequences. The composition may further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0032] In some embodiments, a composition comprises a first engineered extracellular vesicle and a second engineered extracellular vesicle, the first engineered extracellular vesicle comprising a first fusion protein comprising formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; the second engineered extracellular vesicle comprising a second fusion protein comprising the formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; wherein both the first fusion protein and the second fusion protein are displayed on a surface of the first engineered extracellular vesicle and the second engineered extracellular vesicle, respectively; and wherein Li - L4 are optional linker sequences, wherein each of Li - L4 comprise an amino acid sequence (GGGGS)n (SEQ ID NO: 50), wherein n is 1, 2, 3, 4, or 5.

[0033] The disclosure also provides for methods of treating acute myeloid leukemia (AML) comprising the steps of administering to a subject in need thereof an effective amount of any one of the engineered extracellular vesicles or a composition thereof, wherein the engineered extracellular vesicle or the composition treats the AML. In some embodiments, the effective amount is about 0.1 mg / kg to about 30 mg / kg.

[0034] These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

[0035] 4

[0036] 530.045W01

[0037] 2024-123-02 BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0039] FIG. 1A-D. Design, generation, and characterization of aCD3-aCLL-l Exos. (A) Schematic of aCD3-aCLL-l Exos with PDFGR TMD-mediated surface display of aCD3 and aCLL-1 scFv. A HA tag was placed at N-terminus of the fusion protein. (B) Immunoblot analysis of purified exosomes. (C) Size distribution of aCD3-aCLL-l Exos (D) Flow cytometric analysis of binding to U937 (CLL-1+++), HL60 (CLL-1++), KG1A (CLL-F) and Jurkat (CD3+) cells, a = antiCD3-antiCLL-l exosomes, b = antiCLL-1 exosomes, c = antiCD3 exosomes, and d = native exosomes.

[0040] FIG. 2A-C. In vitro biological activity of aCD3 -aCLL-1 Exos. (A) In vitro cytotoxicity of aCD3 -aCLL-1 Exos for U937 (CLL-1+++) (circle), HL60 (CLL-1++) (square), KG1A (CLL-F) (diamond) AML cell lines. Non-activated human PBMCs (effector cells(E)) were incubated with AML cells (target cells (T)) at an E: T ratio of 8 for 24 hours in the presence of aCD3-aCLL-l Exos, followed by measurements of viabilities of target cells. (B) and (C) / / / vitro U937-dependent T-cell activation by aCD3-aCLL-l Exos as evaluated by percentages of CD69+T cells (B) and secreted IFN-y (C). Non-activated human PBMCs were incubated without or with AML cells at an E: T ratio of 2 for 24 hours in the presence of native exosomes (circle), a mixture (1:1) of aCD3 Exos and aCLL-1 Exos (square), or aCD3-aCLL-l Exos (triangle), followed by measurements of percentages of CD69+T cells and levels of secreted IFN-y.

[0041] FIG. 3A-E. Design, generation, and characterization of PD-1-CD70 Exos. (A) Schematic of PD-1-CD70 Exos with surface displayed PD-1 and CD70 via a CD9-based fusion protein. The CD9 fusion includes an N-terminal HA tag and C-terminal His6tag. (B) Immunoblots of native exosomes and PD-1-CD70 Exos. (C) Size distribution of PD-1-CD70 Exos. (D) Sandwich ELISA analysis of binding of PD-1-CD70 Exos to PD-L1 and CD27. His6-tagged CD27 and recombinant human PD-L1 were used as capture and detection reagent, respectively. (E) Dose-dependent activation of T cells by PD-1-CD70 Exos as evaluated by

[0042] 5

[0043] 530.045W01

[0044] 2024-123-02 secreted IFN-y. The levels of secreted IFN-y were measured by ELISA. Circle = native exosomes, square = PD-1-CD70 exosomes.

[0045] FIG. 4A-G. Generation and characterization of aCD3-aCLL-l-PD-l-CD70 PRIME Exos. (A) Schematic of aCD3-aCLL-l-PD-l-CD70 PRIME Exos with genetically displayed aCD3 scFv, aCLL-1 scFv, PD-1, and CD70. (B) Immunoblots of PRIME Exos. Native exosomes, aCD3 -aCLL-1 Exos, PD-1-CD70 Exos, and a mixture (1: 1) of aCD3 -aCLL-1 Exos and PD-1-CD70 Exos were included for comparison. (C) Size distribution of PRIME Exos. (D) and (E) ELISA analysis of binding of PRIME Exos to PD-L1 (D), and CD27 (E). (F) and (G) Flow cytometric analysis of binding of PRIME-Exos to IFN-y-stimulated U937 cells (CLL-1+PD-L1+) (F) and Jurkat cells (CD3+) (G). For 4D and 4E, down triangle = PRIME exosomes, square = PD-1-CD70 exosomes, circle = native exosomes. For 4F and 4G, a = PRIME exosomes, b = PD-1-CD70 exosomes, c = anti CD3 -anti CLL-1 exosomes, d = native exosomes, and d = control.

[0046] FIG. 5A-C. In vitro biological activity of aCD3-aCLL-l-PD-l-CD70 PRIME Exos. (A) In vitro cytotoxicity of PRIME Exos for U937 cells. Non-activated human PBMCs were incubated with U937 cells at an E: T ratio of 8 for 24 hours in the presence of native exosomes (circle), aCD3-aCLL-l Exos (square), PD-1-CD70 Exos (triangle), a mixture (1:1) of aCD3-aCLL-1 Exos and PD-1-CD70 Exos (upside down triangle), or PRIME Exos (diamond), followed by determination of cell viability. (B) and (C) Time-dependent T-cell activation for HL60 (CLL-1+) (B) and KG1A (CLL-1') (C) cells by PRIME Exos. Non-activated human PBMCs were incubated with (B) HL60 or (C) KG1 A cells at an E: T ratio of 2 for 24-96 hour in the absence or presence of native exosomes, aCD3 -aCLL-1 Exos, PD-1-CD70 Exos, a mixture (1:1) of aCD3-aCLL-l Exos and PD-1-CD70 Exos, or PRIME Exos, followed by measurement of secreted IFN-y. For 5B an 5C, closed circle = control, square = native exosomes, triangle = antiCD3 -anti CLL-1 exosomes, down triangle = PD-l=CD70 exosomes, diamond = a mixture (1: 1) of aCD3-aCLL-l Exos and PD-1-CD70 Exos, open circle = PRIME exosomes.

[0047] FIG. 6A-G. In vivo efficacy of aCD3-aCLL-l-PD-l-CD70 PRIME Exos. (A) IVIS images of mice in different groups post inoculation of luciferase-expressing U937 cells. Female NSG mice (n=5) were inoculated with U937 cells via tail vein injections. Human PBMCs from the same healthy donor were intraperitoneally injected into mice on day 1 and 7 after tumor inoculation. Beginning on day 2, mice were administered with PBS or various types of exosomes (10 mg kg'1for monotherapy and 20 mg kg'1for combination therapy) every other day for six times via intravenous injections. (B) Luminescence signals for mice in different

[0048] 6

[0049] 530.045W01

[0050] 2024-123-02 groups following injection of U937 cells. (C) Average body weights of mice during the efficacy study. (D) The weights of collected spleen samples. (E)-(G) Percentages of the mCD45 hCLL- 1+cell population in total living cells of blood (E), spleen (F), and bone marrow (BM) (G) on day 15. Closed circle=PBS; square = native exos; triangle = antiCD3-antiCLL-l exos; upside down triangle = PD-1-CD70 exos; diamond = anti CD3 -anti CLL-1 + PD-1-CD70 exos; open circle = PRIME exos. The furthest right group of circles in each graph are open circles.

[0051] FIG. 7A-B. In vivo safety profile of aCD3-aCLL-l-PD-l-CD70 PRIME Exos. (A) and (B) Creatinine concentrations (A) and ALT activities (B) in plasma at the end of study. Closed circle=PBS; square = native exos; triangle = antiCD3 -anti CLL-1 exos; upside down triangle = PD-1-CD70 exos; diamond = anti CD3 -anti CLL-1 + PD-1-CD70 exos; open circle = PRIME exos. The furthest right group of circles in each graph is open circles.

[0052] FIG. 8A-F. In vivo T-lymphocyte infiltration by aCD3-aCLL-l-PD-l-CD70 PRIME Exos. (A)-(C) Percentages of CD8+T cells in CD45+cells in blood (A), spleen (B), and bone marrow (BM) (C) on day 15. (D)-(F) Percentages of CD4+CD25+Foxp3+Tregs in CD45+cells in blood (D), spleen (E), and BM (F) at the end of study. Open circle=PBS; square = native exos; triangle = anti CD3 -anti CLL-1 exos; upside down triangle = PD-1-CD70 exos; diamond = anti CD3 -anti CLL-1 + PD-1-CD70 exos; closed circle = PRIME exos. The furthest right group of circles in each graph is open circles.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] Definitions.

[0055] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley ’s Condensed Chemical Dictionary 14thEdition, by R. I. Lewis, John Wiley & Sons, New York, N. Y., 2001 or Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology. Harper Perennial, N. Y. (1991). General laboratory techniques (DNA extraction, RNA extraction, cloning, PCR amplification, cell culturing, etc.) are known in the art and described, for example, in Molecular Cloning: A Laboratory Manual, I. Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, 2012.

[0056] References in the specification to "one embodiment", "an embodiment", etc., indicate 7

[0057] 530.045W01

[0058] 2024-123-02 that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0059] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0060] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five substituents on the ring.

[0061] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0062] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two

[0063] 8

[0064] 530.045W01

[0065] 2024-123-02 integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0066] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0067] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number 2”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5,... 9, 10. It also means 1.0, 1.1, 1.2. 1.3,..., 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers less than number 10, as discussed above. Similarly, if the variable disclosed is a number greater than “number 10”, it implies a

[0068] 9

[0069] 530.045W01

[0070] 2024-123-02 continuous range that includes whole numbers and fractional numbers greater than number 10. These ranges can be modified by the term “about”, whose meaning has been described above.

[0071] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0072] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0073] An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect.

[0074] Alternatively, the terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an agent or a composition or combination of compositions being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations. However, the precise determination of what

[0075] 10

[0076] 530.045W01

[0077] 2024-123-02 would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration of the compositions, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment).

[0078] As used herein, "subject" or “patient” means an individual having symptoms of, or at risk for, a disease or other malignancy. A patient may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the methods provided herein, the mammal is a human.

[0079] The terms “administering” or “administration” in reference to delivering engineered vesicles to a subject include any route of introducing or delivering to a subject the engineered vesicles to perform the intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), intracranially, or topically. Additional routes of administration include intraorbital, infusion, intraarerial, intracapsular, intracardiac, intrademal intrapuhnonary, intraspinal intrastemal, intrathecal intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Administration includes selfadministration and the administration by another. The compounds and compositions described herein may be administered with additional compositions to prolong stability and activity of the compositions, or in combination with other therapeutic drugs.

[0080] The terms "treating", "treat" and "treatment" include (i) inhibiting the disease, pathologic or medical condition or arresting its development; (ii) relieving the disease, pathologic or medical condition; and / or (iii) diminishing symptoms associated with the disease, pathologic or medical condition. As such, the term "treatment" can include medical and therapeutic administration, as appropriate.

[0081] 11

[0082] 530.045W01

[0083] 2024-123-02 The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.

[0084] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation, or limitations not specifically disclosed herein.

[0085] As used herein, the terms “selectively binds to” or “preferentially binds to” mean that the compound, peptide or peptidomimetic, or other agent binds to the indicated molecule(s) or class of molecules with a higher affinity (e.g., at least 10-fold, in certain aspects of the invention: 100-fold) compared to a reference molecule.

[0086] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative

[0087] 12

[0088] 530.045W01

[0089] 2024-123-02 substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif.).

[0090] As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue 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 window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0091] The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. In certain embodiments, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Thus, the invention also provides nucleic acid molecules and peptides that are substantially identical to the nucleic acid molecules and peptides presented herein.

[0092] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0093] Nucleic acid sequences cited herein are written in a 5' to 3' direction unless indicated otherwise. The term "nucleic acid" refers to either DNA or RNA or a modified form thereof comprising the purine or pyrimidine bases present in DNA (adenine " A", cytosine " C", guanine " G", thymine " T") or in RNA (adenine " A", cytosine " C", guanine " G", uracil " U"). Interfering

[0094] 13

[0095] 530.045W01

[0096] 2024-123-02 RNAs provided herein may comprise " T" bases, for example at 3' ends, even though " T" bases do not naturally occur in RNA. In some cases, these bases may appear as "dT" to differentiate deoxyribonucleotides present in a chain of ribonucleotides.

[0097] The term "antibody" as used herein refers to a polypeptide (or set of polyptptides) of the immunoglobulin family that is capable of binding an antigen non- covalently, reversibly and specifically. For example, a naturally occurring "antibody" of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen, which is sometimes referred to herein as the antigen binding domain. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, bispecific or multispecific antibodies and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies described herein), single chain variable fragments, and single domain antibodies. The antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino- terminus of the antibody. The N-terminus is a variable region

[0098] 14

[0099] 530.045W01

[0100] 2024-123-02 and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

[0101] A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances.

[0102] “Plasmids” used in genetic engineering are called “plasmid vectors”. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for.

[0103] As used herein the term “fused” intends conjugated or joined by a chemical bond, for example a covalent bond.

[0104] Aspects of the invention are described, for example, in Zhang et al., Molecular Therapy, Volume 33, Issue 3, 1091 - 1104 (2025) and supplemental information, U. S. Patent No. 11,938,219 to Zhang etal., and U. S. Patent Publication No. 2025 / 0092143 to Zhang etal., and are incorporated herein by reference in their entirety.

[0105] Embodiments of the Invention.

[0106] This disclosure provides for engineered extracellular vesicles expressing one or more fusion proteins that may be used, for example, to activate and recruit T-cells to kill cancer cells. Embodiments of an engineered extracellular vesicle or compositions thereof may comprise, consist essentially of, or consist of a first fusion protein comprising a formula A-B-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second fusion protein comprising the formula D-E-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; and wherein

[0107] 15

[0108] 530.045W01

[0109] 2024-123-02 both the first fusion protein and the second fusion protein are displayed on a surface of the engineered extracellular vesicle.

[0110] In some embodiments, the anti-CD-3 antibody moiety comprising six complementary determining regions (CDRs), wherein the six CDRs comprise RASQDIRNYLN (SEQ ID NO: 25), YTSRLHS (SEQ ID NO: 26), QQGNTLPWT (SEQ ID NO: 27), GYTMN (SEQ ID NO: 28), LINPYKGVSTYNQKFKD (SEQ ID NO: 29), and SGYYGDSDWYFDV (SEQ ID NO: 30), and the anti-CLL-1 antibody moiety comprises six complementary determining regions (CDRs), wherein the six CDRs comprise RASSNVISSYVH (SEQ ID NO: 31), STSNLAS (SEQ ID NO: 32), QQYSGYPLT (SEQ ID NO: 33), SAYYWN (SEQ ID NO: 34), YISYDGRNNYNPSLKN (SEQ ID NO: 35), and EGDYDVGNYYAMDY (SEQ ID NO: 36).

[0111] In other embodiments, an engineered extracellular vesicle may comprise a first fusion protein comprising consecutive amino acids, beginning from the amino terminus of the first fusion protein according to formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second fusion protein comprising consecutive amino acids, beginning from the amino terminus of the second fusion protein according to formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; wherein both the first fusion protein and the second fusion protein are displayed on a surface of the engineered extracellular vesicle; and wherein L1 – L4 are optional linker sequences.

[0112] In some embodiments, an engineered extracellular vesicle may comprise a first fusion protein comprising consecutive amino acids, beginning from the amino terminus of the first fusion protein according to formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second fusion protein comprising consecutive amino acids, beginning from the amino terminus of the second fusion protein according to formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; wherein both the first fusion protein and the second fusion protein are displayed on a surface of the engineered extracellular vesicle; wherein the anti-CD-3 antibody moiety comprising six complementary determining regions (CDRs), wherein the six CDRs comprise RASQDIRNYLN (SEQ ID NO: 25), YTSRLHS (SEQ ID NO: 26), QQGNTLPWT (SEQ ID NO: 27), GYTMN (SEQ ID NO: 28), LINPYKGVSTYNQKFKD (SEQ ID NO: 29), and SGYYGDSDWYFDV (SEQ ID NO: 30), and the anti-CLL-1 antibody moiety comprises six complementary determining regions (CDRs), wherein the six CDRs comprise RASSNVISSYVH (SEQ ID NO: 31), STSNLAS (SEQ ID NO: 32), QQYSGYPLT (SEQ ID NO: 33), SAYYWN (SEQ ID NO: 34),

[0113] 16

[0114] 530.045W01

[0115] 2024-123-02 YISYDGRNNYNPSLKN (SEQ ID NO: 35), and EGDYDVGNYYAMDY (SEQ ID NO: 36); and wherein L1 – L4 are optional linker sequences.

[0116] In some embodiments, two distinct populations of extracellular vesicles each include only one of the first fusion protein or the second fusion protein. For example, a first engineered extracellular vesicle comprises a first fusion protein comprising formula A-B-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second engineered extracellular vesicle comprises a second fusion protein comprising the formula D-E-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; and wherein both the first fusion protein and the second fusion protein are displayed on a surface of the first engineered extracellular vesicle and the second engineered extracellular vesicle, respectively.

[0117] In some embodiments, a first engineered extracellular vesicle comprises a first fusion protein comprising formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second engineered extracellular vesicle comprises a second fusion protein comprising the formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; wherein both the first fusion protein and the second fusion protein are displayed on a surface of the first engineered extracellular vesicle and the second engineered extracellular vesicle, respectively; and wherein Li - L4 comprise the amino acid sequence (GGGGS)n (SEQ ID NO: 50), wherein n is 1, 2, 3, 4, or 5.

[0118] In some embodiments, a engineered extracellular vesicle may comprise a first fusion protein comprising consecutive amino acids, beginning from the amino terminus of the first fusion protein according to formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second engineered extracellular vesicle comprises a second fusion protein comprising consecutive amino acids, beginning from the amino terminus of the second fusion protein according to formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; wherein both the first fusion protein and the second fusion protein are displayed on a surface of the respective engineered extracellular vesicle; wherein the anti-CD-3 antibody moiety comprising six complementary determining regions (CDRs), wherein the six CDRs comprise RASQDIRNYLN (SEQ ID NO: 25), YTSRLHS (SEQ ID NO: 26), QQGNTLPWT (SEQ ID NO: 27), GYTMN (SEQ ID NO: 28), LINPYKGVSTYNQKFKD (SEQ ID NO: 29), and SGYYGDSDWYFDV (SEQ ID NO: 30), and the anti-CLL-1 antibody moiety comprises six complementary determining regions (CDRs), wherein the six CDRs

[0119] 17

[0120] 530.045W01

[0121] 2024-123-02 comprise RASSNVISSYVH (SEQ ID NO: 31), STSNLAS (SEQ ID NO: 32), QQYSGYPLT (SEQ ID NO: 33), SAYYWN (SEQ ID NO: 34), YISYDGRNNYNPSLKN (SEQ ID NO: 35), and EGDYDVGNYYAMDY (SEQ ID NO: 36); and wherein L1 – L4 are optional linker sequences.

[0122] In some embodiments, the first and / or second fusion protein further comprises a signal peptide on the N-terminus of the fusion protein. An exemplary signal peptide includes METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 47).

[0123] In some embodiments, the anti-CD-3 antibody moiety is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 3. In some embodiments, the anti-CLL-1 antibody moiety is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 4.

[0124] In some embodiments of the engineered extracellular vesicle, each of the first antibody moiety and the second antibody moiety comprise a single chain variable fragment (scFv), a single domain antibody, a bispecific antibody, or a multispecific antibody.

[0125] Preferably, both the first antibody moiety and the second antibody moiety are scFvs. The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VHor may comprise VH-linker-VL. ScFv molecules are known in the art and their production is described, for example, in U. S. Patent No. 4,946,778, and U. S. Patent No. 5,641,870.

[0126] In other embodiments, the first antibody moiety and / or the second antibody moiety are bispecific antibodies. The term “bispecific antibody” refers to an antibody that shows specificities to two different types of antigens. In other embodiments, the first antibody binding moiety and / or the second antibody binding moiety are multispecific antibodies. The term

[0127] 18

[0128] 530.045W01

[0129] 2024-123-02 "multispecific antibody" as used herein refers to a molecule that binds to two or more different epitopes on one antigen or on two or more different antigens. Recognition of each antigen is generally accomplished with an "antigen binding domain". The multispecific antibody may include one polypeptide chain that comprises a plurality, e.g., two or more, e.g., two, antigen binding domains. In some embodiments, the multispecific antibody may include two, three, four or more polypeptide chains that together comprise a plurality, e.g., two or more, e.g., two, antigen binding domains. Examples of the production and isolation of bispecific and multispecific antibodies are described in, for example, PCT Publication Nos. W02014 / 031174 and W02009 / 080252.

[0130] Also contemplated are single domain antibodies. The term “single domain antibodies” refers to the variable regions of either the heavy (VH) or light (VL) chain of an antibody. Single domain antibodies are described, for example in U. S. Patent Publication No. 2006 / 0002935.

[0131] In some embodiments, the anti-CD-3 antibody moiety is a scFv comprising an amino acid sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 3. In some embodiments, the anti-CLL-1 antibody moiety is a scFv comprising an amino acid sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 4.

[0132] In some embodiments, the anti-CD-3 antibody moiety is a scFv comprising a DNA sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 19. In some embodiments, the anti-CLL-1 antibody moiety is a scFv comprising a DNA sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 20.

[0133] In some embodiments, the first and second antibody moieties are anchored to and displayed on the surface of an extracellular vesicle such as an exosome. In some embodiments,

[0134] 19

[0135] 530.045W01

[0136] 2024-123-02 the first a second antibody moieties are fused to a portion of an exosomal membrane protein. In some embodiments, the portion of an exosomal membrane protein is a transmembrane domain of plate derived growth factor receptor, or PDGFR. In other embodiments, the portion of an exosomal membrane protein is a transmembrane domain of the CD9 protein. Preferably, the PDGFR and the CD9 protein are human PDGFR and human CD9. In some embodiments, the PDGFR TMD is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 7. In some embodiments, the CD9 TMD is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 8.

[0137] In certain embodiments, the scFv molecules may be produced from cDNA molecules or other polynucleotides encoding the variable regions of the heavy and light chains of the mAb that may be amplified by standard polymerase chain reaction (PCR) methodology using a set of primers for immunoglobulin heavy and light variable regions (Clackson (1991) Nature, 352, 624-628) (Also see U. S. Patent No.6, 287, 569 to Kipps etal.). The amplified cDNAs encoding mAb heavy and light chain variable regions then may be linked together with a linker polypeptide in order to generate a recombinant scFv DNA molecule. Other polynucleotide elements may be included in the recombinant fusion protein such as an epitope tag and / or another protein that may anchor the scFv molecule on the surface of the exosome. In certain embodiments, the scFv molecules are genetically fused to the polynucleotide sequence of a hemagglutinin epitope tag and a transmembrane segment of PDGFR.

[0138] In some embodiments, the first fusion protein comprises a first and second antibody moiety that binds separately to a T-cell marker protein and a cancer cell surface-marker protein, respectively. In some embodiments, the T-cell marker protein comprises CD3and the cancer cell surface-marker protein comprises CLL-1. In some embodiments, the first fusion protein comprises an anti-CD3 scFv, a transmembrane domain, and an anti-CLL-1 scFv.

[0139] In some embodiments, the second fusion protein comprises a PD-1 protein or portions thereof, a transmembrane domain, and a CD70 protein or portions thereof. In some embodiments, the PD-1 protein comprises an amino acid sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical,

[0140] 20

[0141] 530.045W01

[0142] 2024-123-02 about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 5. In some embodiments, the CD70 protein comprises an amino acid sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 6.

[0143] In some embodiments, the PD-1 protein comprises a DNA sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 99% identical, or identical to SEQ ID NO: 22. In some embodiments, the CD70 protein comprises a DNA sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 86% identical, about 87% identical, about 88% identical, about 89% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 23.

[0144] Peptide linker groups may be used to connect various portions of the fusion proteins, for example, between an scFv and the PDGFR transmembrane domain. In other embodiments, additional linker proteins may be positioned between the transmembrane domain (e.g., of CD9) and each of the flanking protein moieties. For example, in some embodiments, flexible (GGGGS)₂ (SEQ ID NO: 53) linkers are disposed between the CD9 transmembrane protein and each of the flanking protein moieties or between each of the antibody moieties and the PDGFR transmembrane protein.

[0145] In other embodiments, the linker sequence is either (GGGS)n (SEQ ID NO: 48) where n is an integer from 1 to 5 (i.e., 1, 2, 3, 4, 5) or (GGGGS)n (SEQ ID NO: 50) where n is an integer from 1 to 5. In some embodiments, the(GGGS)n (SEQ ID NO: 48) linker sequence is a (GGGS)4 (SEQ ID NO: 51) peptide and the (GGGGS)n (SEQ ID NO: 50) linker sequence is a (GGGGS)₅ (SEQ ID NO: 52) peptide. In some embodiments, the(GGGS)n (SEQ ID NO: 48) linker sequence is a (GGGS)2 (SEQ ID NO: 49) peptide The linker sequence may be varied depending on the polypeptide portions to be linked to form the fusion protein. Further linker examples include poly(L-Gly), (Poly L-Glycine linkers); poly(L-Glu), (PolyL-Glutamine linkers); poly (1-Lys), (Poly L-Lysine linkers).

[0146] Each fusion protein also may include one or more epitope tags, affinity tags, solubility enhancing tags, and the like. Examples of various additional tags and linkers that may be used with the present invention include, haemagglutinin (HA) epitope, myc epitope, histidine tag,

[0147] 21

[0148] 530.045W01

[0149] 2024-123-02 chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), calmodulin binding peptide, biotin carboxyl carrier protein (BCCP), FLAG octapeptide, nus, green fluorescent protein (GFP), thioredoxin, poly(NANP), V5, S-protein, streptavidin, SBP, poly(Arg), DsbA, c-myc-tag, HAT, cellulose binding domain, softag 1, softag3, small ubiquitin-like modifier (SUMO), and ubiquitin (Lib). In certain embodiments, the fusion protein includes an epitope tag at the n-terminus or the c-terminus of the fusion protein. In preferred embodiments, the epitope tag is a hemagglutinin (HA) epitope tag YPYDVPDYA disposed at the N-terminus of the fusion protein, and / or a 6x Histidine tag at the C-terminal end of the fusion protein.

[0150] In some embodiments, the first fusion protein may have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO: 1 or SEQ ID NO: 13. In some embodiments, a linker (GGGS)n (SEQ ID NO: 48) or (GGGGS)n (SEQ ID NO: 50) may be inserted after amino acid position 518 of SEQ ID NO: 13, where n is 1, 2, 3, 4, or 5. In some embodiments, the first fusion protein may comprise the formula T1-A-L1-B-L2-T2-C, where Ti is an epitope tag, A is anti-CD3 scFv, Li is a first linker sequence, B is anti-CLL-1 scFv, L2 is a second linker sequence, T2 is a second epitope tag, and C is a transmembrane domain. In one certain embodiment, Ti is a hemagglutinin epitope tag, Li is (GGGGS)n (SEQ ID NO: 50), where n is an integer from 1 to 5, L2 is (GGGGS)n (SEQ ID NO: 50), where n is an integer from 1 to 5, and T2 is a Myc epitope tag.

[0151] In some embodiments, the second fusion protein may have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO: 2 or SEQ ID NO: 14. In some embodiments, the second fusion protein may comprise the formula T1-D-L3-E-L4 -F-T2, where Ti is an epitope tag, D is PD-1 protein, L3 is a third linker sequence, E is CD9 protein (e.g., a TMD of CD9), L4 is a fourth linker sequence, T2 is a second epitope tag, and F is CD70 protein. In one certain embodiment, Ti is a hemagglutinin epitope tag, L3 is (GGGGS)n (SEQ ID NO: 50), where n is an integer from 1 to 5, L4 is (GGGGS)n (SEQ ID NO: 50), where n is an integer from 1 to 5, and T2 is a 6x histidine epitope tag.

[0152] 22

[0153] 530.045W01

[0154] 2024-123-02 In some embodiments, the first fusion protein comprises an amino acid sequence that is at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO: 1 or SEQ ID NO: 13. In some embodiments, the first fusion protein comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 1 or SEQ ID NO: 13. In some embodiments, the first fusion protein comprises an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 13.

[0155] In some embodiments, the first fusion protein comprises an amino acid sequence that is at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO: 2 or SEQ ID NO: 14. In some embodiments, the first fusion protein comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 2 or SEQ ID NO: 14. In some embodiments, the first fusion protein comprises an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 14.

[0156] Embodiments of an engineered extracellular vesicle or compositions thereof may comprise, consist essentially of, or consist of a first fusion protein comprising a formula A-B-C, wherein A is an anti-CD3 antibody moiety, wherein A is about 85% to 100% identical to SEQ ID NO: 3, B is an anti-CLL-1 antibody moiety, wherein B is about 85% to 100% identical to SEQ ID NO: 4, and C is a first transmembrane domain that is about 85% to 100% identical to SEQ ID NO: 7; a second fusion protein comprising the formula D-E-F, wherein D is a PD-1 protein that is about 85% to 100% identical to SEQ ID NO: 5, E is a second transmembrane domain that is a about 85% to 100% identical to SEQ ID NO: 9, and F is a CD70 protein that is about 85% to 100% identical to SEQ ID NO: 6; and wherein both the first fusion protein, and the second fusion protein are displayed on the surface of the extracellular vesicle.

[0157] Embodiments of an engineered extracellular vesicle or compositions thereof may comprise, consist essentially of, or consist of a first fusion protein comprising a formula A-B-C, wherein A is an anti-CD3 antibody moiety, wherein A is about 95% to 100% identical to SEQ ID NO: 3, B is an anti-CLL-1 antibody moiety, wherein B is about 95% to 100% identical to SEQ ID NO: 4, and C is a first transmembrane domain that is about 95% to 100% identical to SEQ ID NO: 7; a second fusion protein comprising the formula D-E-F, wherein D is a PD-1 protein that is about 95% to 100% identical to SEQ ID NO: 5, E is a second transmembrane domain that is a about 95% to 100% identical to SEQ ID NO: 9, and F is a CD70 protein that is about 95% to 100% identical to SEQ ID NO: 6; and wherein both the first fusion protein, and the second fusion protein are displayed on the surface of the extracellular vesicle.

[0158] 23

[0159] 530.045W01

[0160] 2024-123-02 In some embodiments, the first fusion protein comprises a DNA sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 99% identical, or identical to SEQ ID NO: 15 or 17. In some embodiments, the second fusion protein comprises a DNA sequence that is about 60% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 86% identical, about 87% identical, about 88% identical, about 89% identical, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or identical to SEQ ID NO: 16 or 18.

[0161] Methods of creating fusion proteins are disclosed, for example, in U. S. Pat. No.

[0162] 6,403,769 to LaRochelle et al. and are well known in the art.

[0163] In certain embodiments, the subject fusion proteins may be delivered via an expression construct to cells, including a nucleic acid that provides a coding sequence for a fusion protein. For instance, the expression construct can encode a fusion protein that is secreted in an exosome by the transduced cell.

[0164] As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as appropriate to the context or as applicable to the embodiment being described, both single-stranded polynucleotides (such as antisense) and double-stranded polynucleotides (such as siRNAs).

[0165] A “protein coding sequence” or a sequence that “encodes” a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the ease of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 ' (amino) terminus and a translation stop codon at the 3 ' (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

[0166] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrated vector, or “integrated vector,” which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, e.g., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to

[0167] 24

[0168] 530.045W01

[0169] 2024-123-02 which they are operatively linked are referred to herein as “expression vectors.” In the present specification, “plasmid” and “vector” are used interchangeably unless otherwise clear from the context. In the expression vectors, regulatory elements controlling transcription can be generally derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as retroviruses, adenoviruses, and the like, may be employed.

[0170] Vectors suitable for use in preparation of proteins and / or protein conjugates include those selected from baculovirus, phage, plasmid, phagemid, cosmid, fosmid, bacterial artificial chromosome, viral DNA, Pl-based artificial chromosome, yeast plasmid, and yeast artificial chromosome. For example, the viral DNA vector can be selected from vaccinia, adenovirus, foul pox vims, pseudorabies and a derivative of SV40. Suitable bacterial vectors for use in various methods include pQE70™, pQE60, pQE-9, pBLUESCRIPT SK, pBLUESCRIPT™ KS, pTRC99a™, pKK223-3™, pDRS40™, PAC™ and pR. IT2T™. Suitable eukaryotic vectors for use in various methods include pWLNEO™, pXTI™, pSG5™, pSVK3™, pBPV™, pMSG™, and pSVLSV40™. Suitable eukaryotic vectors for use in various methods include pWLNEO™, pXTI™, pSG5™, pSVK3™, pBPV™, pMSG™, and pSVLSV40™. Those of skill in the art can select a suitable regulatory region to be included in such a vector, for example from lacl, lacZ, T3, T7, apt, lambda PR, PL, trp, CMV immediate early, HSV thymidine kinase, early and late SV40, retroviral LTR, and mouse metallothionein-I regulatory regions.

[0171] Host cells in which the vectors containing the polynucleotides encoding the protein conjugates can be expressed include, for example, a bacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or a plant cell. For example, E. coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g. COS-7), or Bowes melanoma cells are all suitable host cells for use in the methods described herein.

[0172] In some embodiments, the engineered extracellular vesicle is an exosome or other membrane-enclosed bodies such as described in PCT (International) Publication Nos. W02017 / 161010 and WO2016 / 077639, and U. S. Patent Publication Nos. US2016 / 0168572, US2015 / 0290343, and US2007 / 0298118. The extracellular vesicle, e.g., a cell-derived vesicle comprising a membrane that encloses an internal space and has a smaller diameter than the cell from which it is derived may have a diameter from 20 nm to 1000 nm. In some embodiments, the engineered extracellular vesicle comprises an apoptotic body, a fragment of a cell, a vesicle derived from a cell by direct or indirect manipulation, a vesiculated organelle, and a vesicle

[0173] 25

[0174] 530.045W01

[0175] 2024-123-02 produced by a living cell (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane), or an exosome.

[0176] In embodiments, the engineered extracellular vesicle comprises an exosome. In some embodiments, the exosome is a cell-derived small (e.g., between 20-300 nm in diameter, or 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. In embodiments, production of exosomes does not result in the destruction of the source cell. In embodiments, the exosome comprises lipid or fatty acid and polypeptide.

[0177] Populations of engineered extracellular vesicles (e.g., exosomes and / or microvesicles) of the present disclosure can be isolated using any method known by those in the art. Nonlimiting examples include differential centrifugation by ultracentrifugation (Thery et al. (2006) Curr. Protoc. Cell Biol. 30:3.22.1-3.22.29; Witmer et al. (2013) J. Extracellular v.2), sucrose gradient purification (Escola et al. (1998) J. Biol. Chem. 273:20121-20127), and combination filtration / concentration (Lamparski et al. (2002) J. Immunol. Methods 270:211-226).

[0178] After isolation, the cell-derived vesicles, e.g., exosomes can be concentrated to provide a purified population of cell-derived vesicles. Any appropriate method can be used to concentrate the cell-derived vesicles, e.g., exosomes. Non-limiting examples of such include centrifugation, ultrafiltration, filtration, differential centrifugation and column filtration. Further sub-populations can be isolated using antibodies or other agents that are specific for a specific marker expressed by the desired exosome population. Alternatively, shed exosomes may be purified using commercially available extraction kits such as ExoQuick™ and Total Exosome Isolation™.

[0179] The engineered extracellular vesicles can be made from several different types of lipids, e.g., amphipathic lipids, such as phospholipids. The engineered extracellular vesicle may comprise a lipid bilayer as the outermost surface. This bilayer may be comprised of one or more lipids of the same or different type. Examples include, but are not limited to, phospholipids such as phosphocholines and phosphoinositols. Specific examples include without limitation DMPC, DOPC, and DSPC.

[0180] An engineered extracellular vesicle may be mainly comprised of natural phospholipids and lipids such as l,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside. In embodiments, an engineered extracellular vesicle comprises only phospholipids and is less stable in plasma. However, manipulation of the lipid membrane with cholesterol can, in embodiments, increase stability

[0181] 26

[0182] 530.045W01

[0183] 2024-123-02 and reduce rapid release of the encapsulated bioactive compound into the plasma. In some embodiments, the engineered extracellular vesicle comprises l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), e.g., to increase stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679 for review).

[0184] In some embodiments, engineered extracellular vesicles comprise or are enriched for lipids that affect membrane curvature (see, e.g., Thiam et al., Nature Reviews Molecular Cell Biology, 14(12): 775-785, 2013). Some lipids have a small hydrophilic head group and large hydrophobic tails, which facilitate the formation of a fusion pore by concentrating in a local region. In some embodiments, engineered extracellular vesicles comprise or are enriched for negative-curvature lipids, such as cholesterol, phosphatidylethanolamine (PE), diglyceride (DAG), phosphatidic acid (PA), fatty acid (FA). In some embodiments, engineered extracellular vesicles do not comprise, are depleted of, or have few positive-curvature lipids, such as lysophosphatidylcholine (LPC), phosphatidylinositol (Ptdins), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), monoacylglycerol (MAG).

[0185] In some embodiments, the lipids are added to a source cell to produce an engineered extracelluar vesicle. In some embodiments, the lipids are added to source cells in culture which incorporate the lipids into their membranes prior to or during the formation of an engineered extracellular vesicle. In some embodiments, the lipids are added to the cells or engineered extracellular vesicle in the form of a liposome. In some embodiments, methyl-betacyclodextrane (mP-CD) is used to enrich or deplete lipids (see, e.g., Kainu et al, Journal of Lipid Research, 51(12): 3533-3541, 2010).

[0186] In this way, the engineered extracellular vesicles may comprise, for example, DOPE (dioleoylphosphatidylethanolamine), DOTMA, DOTAP, DOTIM, DDAB, alone or together with cholesterol to yield DOPE and cholesterol, DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U. S. Patent No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). Although formation of engineered extracellular vesicles can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679 for review). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al.,

[0187] 27

[0188] 530.045W01

[0189] 2024-123-02 Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.

[0190] In another embodiment of the engineered extracellular vesicles, the lipids may include, but are not limited to, DLin-KC2-DMA4, C12-200 and co-lipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated (see, e.g., Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4) using a spontaneous vesicle formation procedure. Tekmira publications describe various aspects of lipid vesicles and lipid vesicle formulations (see, e.g., U. S. Patent Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943; and 7,838,658 and European Patent Nos. 1766035; 1519714; 1781593, and 1664316), each of which are herein incorporated by reference and may be used and / or adapted to the present invention.

[0191] In some embodiments, an engineered extracellular vesicle described herein may include one or more polymers. The polymers may be biodegradable. Biodegradable polymer vesicles may be synthesized using methods known in the art. Exemplary methods for synthesizing polymer vesicles are described by Bershteyn et al., Soft Matter 4: 1787-1787, 2008 and in U. S. Patent Publication No. 2008 / 0014144, the specific teachings of which relating to microparticle synthesis are incorporated herein by reference.

[0192] Exemplary synthetic polymers which can be used include without limitation aliphatic polyesters, polyethylene glycol (PEG), poly (lactic acid) (PLA), poly (glycolic acid) (PGA), co-polymers of lactic acid and glycolic acid (PLGA), polycarprolactone (PCL), polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone), and natural polymers such as albumin, alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof, including substitutions, additions of chemical groups such as for example alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.

[0193] The disclosure also provides for methods of preparing an extracellular vesicle comprising contacting a cell comprising the extracellular vesicle with an effective amount of a polynucleotide encoding the first and second fusion proteins, and expressing the fusion proteins expressed from the polynucleotide on the surface of the vesicle. For example, the polynucleotide may be about 80%, about 85%, about 90%, about 95, about 99%, or 100 identical to any one of SEQ ID Nos: 15, 16, 17, and 18.

[0194] 28

[0195] 530.045W01

[0196] 2024-123-02 In some embodiments, the engineered extracellular vesicles are formulated into a composition comprising a pharmaceutically acceptable carrier or diluent. In some embodiments, a composition also may include two or more populations of engineered extracellular vesicles, each with a different complement of fusion proteins.

[0197] In some embodiments, the disclosure also provides for a method of treating cancer comprising administering an effective amount of one or more extracellular vesicles as disclosed herein, or a composition thereof, to a subject having or suspected of having cancer whereby the engineered extracellular vesicles selectively activate T-cells to kill the cancer cells.

[0198] The term “cancer,” as used herein, refers to any benign or malignant abnormal growth of cells. Examples include, without limitation, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma. In some embodiments, the cancer is selected from the group of tumor-forming cancers.

[0199] In some embodiments, the disclosure provides for a method of treating acute myeloid leukemia (AML) comprising administering an effective amount of one or more extracellular vesicles as disclosed hereinto a subject having or suspected of having AML whereby the engineered extracellular vesicles selectively activate T-cells to kill AML cancer cells.

[0200] In some embodiments, the disclosure provides for a method of treating acute myeloid leukemia (AML) comprising administering an effective amount of one or more composition as disclosed herein to a subject having or suspected of having AML whereby the engineered extracellular vesicles selectively activate T-cells to kill AML cancer cells.

[0201] 29

[0202] 530.045W01

[0203] 2024-123-02 In some embodiments, an effective amount of an engineered extracellular vesicle as described herein comprises about 0.01 mg / kg to about 150 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 75 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, about 110 mg / kg, about 120 mg / kg, about 130 mg / kg, about 140 mg / kg, or about 150 mg / kg,

[0204] In some embodiments, an effective amount of a composition describe herein comprises about 0.01 mg / kg to about 150 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 75 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, about 110 mg / kg, about 120 mg / kg, about 130 mg / kg, about 140 mg / kg, or about 150 mg / kg,

[0205] Embodiments of the Invention.

[0206] 1. In a first embodiment, an engineered extracellular vesicle comprises a first fusion protein comprising a formula A-B-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second fusion protein comprising a formula D-E-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; wherein both the first fusion protein and the second fusion protein are displayed on a surface of the engineered extracellular vesicle.

[0207] 2. In a second embodiment, a first engineered extracellular vesicle comprises a first fusion protein comprising formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second engineered extracellular vesicle comprises a second fusion protein comprising the formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein;

[0208] 30

[0209] 530.045W01

[0210] 2024-123-02 wherein both the first fusion protein and the second fusion protein are displayed on a surface of the first engineered extracellular vesicle and the second engineered extracellular vesicle, respectively; and wherein L1 – L4 comprise the amino acid sequence (GGGGS)n (SEQ ID NO: 50), wherein n is 1, 2, 3, 4, or 5.

[0211] 3. The engineered extracellular vesicle of embodiment 1 or 2, wherein each of the first antibody moiety and the second antibody moiety comprise a single chain variable fragment (scFv), a single domain antibody, a bispecific antibody, or a multispecific antibody.

[0212] 4. The engineered extracellular vesicle of any one of embodiments 1-3, wherein each of the first antibody moiety and the second antibody moiety comprise a single chain variable fragment (scFv).

[0213] 5. The engineered extracellular vesicle of any one of embodiments 1-4, wherein the first transmembrane domain comprises a transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR) protein.

[0214] 6. The engineered extracellular vesicle of any one of embodiments 1-5, wherein the second transmembrane domain comprises a transmembrane domain of a CD9 protein.

[0215] 7. The engineered extracellular vesicle of any one of embodiments, 1-6, wherein the first fusion protein and the second fusion protein are displayed on the surface of the engineered extracellular vesicle in a 1:1 ratio.

[0216] 8. The engineered extracellular vesicle of any one of embodiments 1-7, wherein the extracellular vesicle comprises one or more of an exosome, a liposome, a microvesicle, an apoptotic body, and a combination thereof.

[0217] 9. The engineered extracellular vesicle of any one of embodiments 1-8, wherein A is an N-terminus and C is the C-terminus of the first fusion protein, or wherein D is the N-terminus and F is the C-terminus of the second fusion protein

[0218] 10. The engineered extracellular vesicle of any one of embodiments 1-9, wherein the engineered extracellular vesicle comprises a particle size of about 25 nm to about 250 nm. 11. The engineered extracellular vesicle of any one of embodiments 1-10, wherein the engineered extracellular vesicle comprises a particle size of about 25 nm to about 150 nm. 12. The engineered extracellular vesicle of any one of claims 1-11, wherein the first fusion protein comprises the formula A-L1-B-L2-C; and the second fusion protein comprising the formula D-L3-E-L4-F; wherein L1 – L4 are optional linker sequences.

[0219] 13. The engineered extracellular vesicle of any one of embodiments 1-12, wherein the linker sequences L1 – L4 comprise the amino acid sequence (GGGGS)n (SEQ ID NO: 50),

[0220] 31

[0221] 530.045W01

[0222] 2024-123-02 wherein n is 1, 2, 3, 4, or 5; or wherein the linker sequences L1 – L4 comprise the amino acid sequence (GGGS)n (SEQ ID NO: 48), wherein n is 1, 2, 3, 4, or 5.

[0223] 14. The engineered extracellular vesicle of embodiments 1-13, wherein at least one of the linker sequences Li - L4 comprises (GGGGS) (SEQ ID NO: 12), (GGGGS)₂ (SEQ ID NO: 53), or (GGGGS)3(SEQ ID NO: 52).

[0224] 15. The engineered extracellular vesicle of any one of embodiments 1-14, wherein the first fusion protein and the second fusion protein further comprise one or more of a hemagglutinin epitope tag, a myc tag, and a 6x histidine epitope tag.

[0225] 16. The engineered extracellular vesicle of any one of embodiments 1-15, wherein the first fusion protein comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 1 or SEQ ID NO: 13; and the second fusion protein comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 2 or SEQ ID NO: 14.

[0226] 17. The engineered extracellular vesicle of any one of embodiments 1-16, wherein the first fusion protein comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1 or SEQ ID NO: 13; and the second fusion protein comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 2 or SEQ ID NO: 14.

[0227] 18. The engineered extracellular vesicle of any one of embodiments 1-17, wherein the first fusion protein comprises an amino acid sequence identical to SEQ ID NO: 1 or SEQ ID NO: 13; and the second fusion protein comprises an amino acid sequence identical to SEQ ID NO: 2 or SEQ ID NO: 14.

[0228] 19. The engineered extracellular vesicle of any one of embodiments 1-18, wherein the first fusion protein comprises a DNA sequence that is at least about 95% identical to SEQ ID NO: 1 or SEQ ID NO: 13; and the second fusion protein comprises a DNA sequence that is at least about 95% identical to SEQ ID NO: 2 or SEQ ID NO: 14.

[0229] 20. The engineered extracellular vesicle of any one of embodiments 1-19, wherein the first fusion protein comprises a DNA sequence identical to SEQ ID NO: 1 or SEQ ID NO: 13; and the second fusion protein comprises a DNA sequence identical to SEQ ID NO: 2 or SEQ ID NO: 14.

[0230] 21. The extracellular vesicle of any one of embodiments 1-20, wherein the anti-CD-3 antibody moiety comprises six complementary determining regions (CDRs), wherein the six CDRs comprise RASQDIRNYLN (SEQ ID NO: 25), YTSRLHS (SEQ ID NO: 26), QQGNTLPWT (SEQ ID NO: 27), GYTMN (SEQ ID NO: 28), LINPYKGVSTYNQKFKD (SEQ ID NO: 29), and SGYYGDSDWYFDV (SEQ ID NO: 30), and the anti-CLL-1 antibody moiety comprises six complementary determining regions (CDRs), wherein the six CDRs

[0231] 32

[0232] 530.045W01

[0233] 2024-123-02 comprise RASSNVISSYVH (SEQ ID NO: 31), STSNLAS (SEQ ID NO: 32), QQYSGYPLT (SEQ ID NO: 33), SAYYWN (SEQ ID NO: 34), YISYDGRNNYNPSLKN (SEQ ID NO: 35), and EGDYDVGNYYAMDY (SEQ ID NO: 36).

[0234] 22. The engineered extracellular vesicle of any one of embodiments 1-21 comprising a first fusion protein comprising consecutive amino acids, beginning from the amino terminus of the first fusion protein according to formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; a second fusion protein comprising consecutive amino acids, beginning from the amino terminus of the second fusion protein according to formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein; wherein both the first fusion protein and the second fusion protein are displayed on a surface of the engineered extracellular vesicle; and wherein L1 – L4 are linker sequences.

[0235] 23. The engineered extracellular vesicle of any one of embodiments 1-22, wherein the first and / or second fusion protein further comprises a signal peptide on the N-terminus of the fusion protein.

[0236] 24. A DNA vector encoding a first fusion protein and second fusion protein, wherein a first DNA sequence encodes the first fusion protein and comprises about 90%, about 95%, about 99% or about 100% identical to SEQ ID NO: 15 or 17, and a second DNA sequence encoding the second fusion protein and comprises about 90%, about 95%, about 99% or about 100% identical to SEQ ID NO: 16 or 18.

[0237] 25. The DNA vector of embodiment 24, wherein each of the first and second fusion proteins are encoded by separate DNA vectors.

[0238] 26. A composition comprising the engineered extracellular vesicle of any one of claims 1-23 and a pharmaceutically acceptable carrier, excipient, or diluent.

[0239] 27. A composition comprising the DNA vector of embodiment 24 or 25, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0240] 28. A composition comprising the extracellular vesicles of embodiment 2 or 21, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0241] 29. A method of treating acute myeloid leukemia (AML) comprising administering to a subject in need thereof an effective amount of the engineered extracellular vesicle of any one of claim 1-23, wherein the engineered extracellular vesicle treats the AML.

[0242] 30. A method of treating acute myeloid leukemia (AML) comprising administering to a subject in need thereof an effective amount of the composition of claim 24 or 25, wherein the composition treats the AML.

[0243] 33

[0244] 530.045W01

[0245] 2024-123-02 31. The method of any one of embodiments 26-28, wherein the effective amount is about 0.1 mg / kg to about 30 mg / kg.

[0246] 32. The method of embodiment 29, wherein the effective amount is about 0.1 mg / kg to about 30 mg / kg.

[0247] Results and Discussion.

[0248] To recruit endogenous T lymphocytes to AML cells, exosomes would need to be reprogrammed with specificity for both cell populations. To this end, we chose to anchor antibodies specific for T-cell CD3 and AML-associated CLL-1 on exosome surfaces via genetic fusion of both single-chain variable fragment (scFv) antibodies in tandem with the transmembrane domain (TMD) of human platelet-derived growth factor receptor (PDGFR) (Figure 1 A). The aCD3 scFv was placed at N-terminus, because this orientation tends to show higher binding affinity to target antigens according to previous studies. In addition to an N-terminal hemagglutinin (HA) epitope tag, a (GGGGS)3 (SEQ ID NO: 52) flexible linker was inserted between scFv antibodies. Fusion constructs expressing aCD3 scFv-PDGFR TMD and aCLL-1 scFv-PDGFR TMD were also generated as controls.

[0249] The designed exosomes were purified through differential centrifugation and ultracentrifugation of harvested media after transient transfection of Expi293F cells with respective expression constructs. Native exosomes were isolated from non-transfected cells. The exosome yields are approximately 200 ug (2 ×1010particles) per 100 mL of cell culture, comparable among different groups. Immunoblot analysis indicated successful expression of designed antibody fusions in exosomes (Figure IB). Nanoparticle tracking analysis (NTA) showed similar size distributions for native exosomes and aCD3-aCLL-l exosomes (aCD3-aCLL-1 Exos) (Figure ID). To validate binding of scFv antibodies toward their cognate antigens, AML cell lines with varied levels of CLL-1 expression including U937(CLL-1+++), HL60 (CLL-1++), and KG1A (CLL-T) along with Jurkat (CD3+) cells were used. Flow cytometry indicated aCD3-aCLL-l Exos can tightly bind to U937, HL60, and Jurkat cells, but not KG1A cells, supporting functional display of scFv antibodies on aCD3-aCLL-l Exo surfaces (Figure 1G).

[0250] Next, we examined in vitro cytotoxicity of aCD3-aCLL-l Exos. It was shown that in the presence of non-activated human peripheral blood mononuclear cells (PBMCs), aCD3-aCLL-1 Exos could potently induce death of U937 and HL60 cells with EC50in a range of 47-149 ng / mL, but little cytotoxic effects on KG1A cells. Native exosomes and the mixture (1:1) of aCD3 Exos and aCLL-1 Exos display no significant cytotoxicity for both cell lines under the same conditions. The potency of aCD3-aCLL-l Exos against AML cells is correlated with 34

[0251] 530.045W01

[0252] 2024-123-02 the expression level of CLL-1 (Figure 2D). In comparison to U937 (CLL-1+++) cells, aCD3-aCLL-1 Exos show decreased cytotoxicity for HL60 (CLL-1++) cells (ECso: 161.2 ± 22.3 ng mL’1) in the presence of PBMCs. Additionally, no cytotoxicity of aCD3-aCLL-l Exos at concentrations up to 50 ug mL’1was observed for HEK293T cells incubated with human PBMCs. These results demonstrate marked and selective killing of CLL-1 -expressing AML cells by aCD3-aCLL-l Exos.

[0253] In vitro T-cell activation by aCD3-aCLL-l Exos was then analyzed by activation markers (CD69 and CD25) and secreted cytokines (interferon-gamma (IFN-y) and interleukin-2 (IL-2)). Flow cytometry indicated that in contrast to native exosomes and the mixture (1:1) of aCD3 Exos and aCLL-1 Exos, aCD3-aCLL-l Exos could induce significant activation of T cells in the presence of CLL-1 -positive U937 cells (Figure 2E). Weak or no T-cell activation was seen for PBMCs treated by exosomes with CLL-1 -negative KG1 A cells. These results are consistent with measured levels of released IFN-y and IL-2 among different groups (Figure 2G), indicating CLL-1 antigen-dependent T-lymphocyte activation by aCD3 -aCLL-1 Exos.

[0254] While exosomes displaying aCD3 and aCLL-1 scFvs can induce CLL-1 -specific immune responses, co-expression of immune modulators on exosomes may enhance and sustain anti-AML immunity through responding to upregulated immune checkpoint pathways. In comparison to HEK293T cells, HL60 and U937 AML cells reveal increased PD-L1 expression upon treatment by IFN-y or aCD3-aCLL-l Exos in the presence of PBMCs. aCD3-aCLL-1 Exos expressing PD-1 molecules can potentially compete for binding to PD-L1, preventing engagement with PD-1 receptors on activated T cells for immune downregulation. Furthermore, aCD3-aCLL-l Exos with displayed CD70 ligands may turn on CD27-mediated signals, augmenting T-cell immunity. To express both PD-1 and CD70 on exosome surfaces, exosomal membrane protein CD9 was genetically fused with N-terminal PD-1 and C-terminal CD70 (Figure 3 A). In addition to an N-terminal HA and C-terminal His6tag, the fusion includes a G4S linker between PD-1 and CD9 and a (G4S)2linker connecting CD9 and CD70.

[0255] The designed PD-1-CD70 exosomes (PD-1-CD70 Exos) were expressed and purified from media of transiently transfected Expi293F cells. Immunoblots showed expression of the PD-1-CD70 fusion protein in exosomes (Figure 3B). NTA indicated comparable size distribution of PD-1-CD70 Exos to those of native exosomes and aCD3-aCLL-l Exos (Figure ID, 11C). Sandwich ELISA revealed dual specificity of PD-1-CD70 Exos for PD-L1 and CD27 (Figure 3D). Flow cytometry further verified that PD-1-CD70 Exos could tightly bind to IFN-y-treated U937 cells (PD-L1+) and CD27-expressing T cells, but not HL60 cells (PD-LF PD-L2‘ CD27. Importantly, in comparison with native exosomes, PD-1-CD70 Exos results in dose-

[0256] 35

[0257] 530.045W01

[0258] 2024-123-02 dependent secretion of IFN-y and IL-2 following incubation with human PBMCs preactivated by aCD3 antibodies (Figure 3H). Collectively, these results support functional display of PD-1 and CD70 immunoregulatory proteins on exosomes.

[0259] With generated functional aCD3-aCLL-l-PDGFR TMD and PD-1-CD9-CD70 fusion proteins, we next sought to produce aCD3-aCLL-l-PD-l-CD70 PRIME Exos by cotransfecting Expi293F cells with both expression constructs (Figure 4A). Immunoblot analysis of purified exosomes indicates stable expression of both fusion proteins in exosomes, similar to those of aCD3-aCLL-l Exos, PD-1-CD70 Exos, and the mixture of aCD3-aCLL-l Exos and PD-1-CD70 Exos (Figure 4B). NTA analysis showed PRIME Exos peaking around 100 nm with a size distribution comparable to those of native exosomes, aCD3-aCLL-l Exos, and PD-1-CD70 Exos (Figure ID, 3C, 4C). The bindings of aCD3 -aCLL-1 -PD-1-CD70 PRIME Exos to recombinant PD-L1, PD-L2, and CD27 were confirmed by ELISA (Figure 4D, 4F). Flow cytometry revealed that PRIME Exos exhibit tighter or comparable binding to IFN-y-treated U937 (CLL-1+++PD-L1+), HL60 (CLL-1++PD-L1 ), and Jurkat (CD3+) cells relative to aCD3-aCLL-l Exos and PD-1-CD70 Exos, but no significant binding to KG1A cells (CLL-1⁻) (Figure 4G). Moreover, aCD3-aCLL-l-PD-l-CD70 PRIME Exos were demonstrated to tightly bind to U937 cells (CLL-1+++) via flow cytometry using both aHA and aHise antibody, supporting co-expression of HA-aCD3-aCLL-l-PDGFR TMD and HA-PD-1-CD9-CD70-Hise fusion proteins on the same membranous vesicle. Collectively, these results show functional display of aCD3 scFv, aCLL-1 scFv, PD-1, and CD70 mol In vitro activity of aCD3-aCLL-l-PD-l-CD70 PRIME Exos was then evaluated. Notably, PRIME Exos display increased cytotoxicity (ECso: 9.7 ± 1.5 ng mL’1) against U937 cells (CLL-1+++) in the presence of non-activated PBMCs compared with aCD3-aCLL-l Exos (ECso: 45.1 ± 6.1 ng mL’1) and the combination of aCD3-aCLL-l Exos and PD-1-CD70 Exos (ECso: 21.3 ± 2.2 ng mL’1) (Figure 5 A). Moreover, PRIME Exos result in higher and more sustainable levels of IFN-y and IL-2 release than aCD3 -aCLL-1 Exos and the combination of aCD3 -aCLL-1 Exos and PD-1-CD70 Exos following incubation with CLL-1 -positive AML cells (HL60 and U937) and nonactivated PBMCs (Figure 5B). In contrast, PBMCs incubated with CLL-1 -negative KG1A cells show no elevated secretion of IFN-y and IL-2 upon exosome treatments (Figure 5F). These results support improved potency for aCD3-aCLL-l-PD-l-CD70 PRIME Exos in promoting anti-AML immunoactivity.

[0260] To probe in vivo efficacy of aCD3-aCLL-l-PD-l-CD70 PRIME Exos, an AML mouse xenograft model was used by inoculating firefly luciferase-expressing U937 cells into NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice via tail vein injections. Mice were administered

[0261] 36

[0262] 530.045W01

[0263] 2024-123-02 with PBS vehicle or different forms of exosomes one day after grafting human PBMCs. Unlike PBS- or native exosome-treated mice with rapidly expanded AML, mice treated by aCD3-aCLL-1 Exos, PD-1-CD70 Exos, the combination (1:1) of aCD3-aCLL-l Exos and PD-1-CD70 Exos, or PRIME Exos exhibit notable inhibition against expansion of the engrafted U937 cells (Figure 6A, 6B). No negative impact on body weights of animals was seen for all treatment groups during the study (Figure 6D). No significant changes were observed for weight ratios of major organs from all animal groups at the end of study, except for spleen and liver. Mice treated with PRIME Exos or the combination of aCD3-aCLL-l Exos and PD-1-CD70 Exos were characterized by reduced weights and / or sizes of liver and spleen, consistent with their efficacy as analyzed by in vivo imaging system (IVIS) imaging and flow cytometry (Figure 6A, 6B, 6H-6J). Among all treatment groups, PRIME Exos along with the combination of aCD3-aCLL-l Exos and PD-1-CD70 Exos show more significant efficacy in suppressing AML development in animals.

[0264] Potential toxicities of exosome treatments were also assessed for the in vivo efficacy study. Determined levels of alanine aminotransferase (ALT) activities (a liver injury marker) and creatinine concentrations (a kidney injury marker) in plasma at the end of study revealed no significant differences (Figure 7A, 7B). Percentages of mouse CD34+CD45+cells in blood, spleen, and bone marrow remained at comparable levels among all groups. Moreover, safety assessments with healthy mice indicated that in comparison with PBS vehicle, aCD3-aCLL-l-PD-1-CD70 PRIME Exos treatment results in no significant changes to body weights, major organ weight ratios, creatinine concentrations and ALT activities in plasma, and levels of CD34+CD45+cells in blood, spleen, and bone marrow. Taken together, these results support great efficacy and safety of PRIME Exos for controlling AML in rodent models

[0265] T lymphocyte infiltration was analyzed for blood, spleen, and bone marrow samples collected at the end of the study. Compared with PBS- or native exosome-treated groups, mice receiving PRIME Exos showed significantly increased CD8+ T cells in the blood, spleen, and bone marrow (Figures 8A-8C). Moreover, significant reduction of regulatory T (Treg) cells in the blood, spleen, and bone marrow was observed in mice administered PRIME Exos (Figures 8D-8F). Consistent with anti-tumor efficacy results, PRIME Exo-treated mice presented the most pronounced increases in CD8+ T cells and CD8+ T cell / Treg cell ratios and decreases in Treg cells in the blood, spleen, and bone marrow among all treatment groups. In addition, CD4+ T cells were slightly reduced in the spleen and bone marrow in mice treated with PRIME Exos. Together, these results suggest that aCD3-aCLL-l-PD-l-CD70 PRIME Exos induce

[0266] 37

[0267] 530.045W01

[0268] 2024-123-02 superb anti-AML effects by fostering cytotoxic CD8+ T cells and diminishing immunosuppressive Treg cells

[0269] Cell-derived exosomes provide a potentially biocompatible, safe, and efficacious candidate modality for therapeutic development. Their spherical membranes support simultaneous expression of multivalent targeting moieties and signaling molecules, promoting tissue specificity and synergistic effects. Herein we utilized genetic approaches to generate aCD3-aCLL-l-PD-l-CD70 PRIME Exos that feature T-cell- and AML-targeting antibodies as well as immune checkpoint modulators. Using preclinical models of AML, we demonstrate potent and CLL-1 -specific activity of PRIME Exos in activating T-cell immunity. Unlike conventional chemotherapeutics, targeted therapies, or immune checkpoint inhibitors, PRIME Exos can not only target CLL-1 -positive AML cells for immune activation, but also respond to possible immune downregulation through acting on distinct immune checkpoint pathways encoded in T cells. Such a tumor-specific, multitargeting strategy may lead to safer and more efficacious immunotherapy for AML.

[0270] Simultaneously targeting T-cell CD3 and AML-cell CLL-1, aCD3-aCLL-l Exos can redirect and activate T cells to induce considerable anti-cancer immunity, in a similar manner to bispecific antibodies. Upon stimulation by pro-inflammatory cytokines, tumor cells at the microenvironment tend to produce immunosuppressive molecules, such as PD-L1 / L2. Codisplay of PD-1 and immunostimulatory CD70 on aCD3-aCLL-l Exos is shown to enhance immune responses through blocking interactions of PD-L1 / L2 with T-cell PD-1 receptors as well as turning on CD27-mediated costimulatory signals, demonstrating therapeutic benefits of PRIME Exos over dual targeting exosomes or antibodies. Moreover, in comparison with the combination of aCD3-aCLL-l Exos and PD-1-CD70 Exos, PRIME Exos reveal improved in vitro and in vivo efficacy, suggesting their capability in promoting synergy between different immunomodulatory pathways.

[0271] While co-transfection of two fusion protein constructs can produce PRIME Exos with all functional domains displayed on the same vesicle, a single vector or stable cell line expressing all designed fusions would improve homogeneity of generated PRIME Exos. Using AML mouse xenograft models, we demonstrate efficacy and safety of PRIME Exos. Nonetheless, further studies for efficacy and toxicity with other AML samples and models are required. In addition, the potential of therapeutic delivery for PRIME Exos could be explored.

[0272] In conclusion, cell-derived exosomes were genetically programmed with antibodies specific for AML and T cells and immune checkpoint molecules. The resulting aCD3-aCLL-

[0273] 38

[0274] 530.045W01

[0275] 2024-123-02 1-PD-1-CD70 PRIME Exos can sustainably induce anti-cancer immunity, representing an immunotherapeutic candidate for AML treatment.

[0276] Pharmaceutical Formulations.

[0277] The compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient, or carrier. The compounds may be added to a carrier in the form of a salt or solvate. For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and [3-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

[0278] Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.

[0279] The compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.

[0280] The compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, compounds can be enclosed in hard or soft-shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet. Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations typically contain at least 0.1% of active compound. The percentage of the compositions and preparations can vary and may conveniently be from about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10%, of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions can be such that an effective dosage level can be obtained.

[0281] 39

[0282] 530.045W01

[0283] 2024-123-02 The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate. A sweetening agent such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0284] The active compound may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.

[0285] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride.

[0286] 40

[0287] 530.045W01

[0288] 2024-123-02 Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and / or gelatin.

[0289] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, optionally followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the solution.

[0290] For topical administration, compounds may be applied in pure form, e.g., when they are liquids. However, it will generally be desirable to administer the active agent to the skin as a composition or formulation, for example, in combination with a dermatologically acceptable carrier, which may be a solid, a liquid, a gel, or the like.

[0291] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol / glycol blends, in which a compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

[0292] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0293] Examples of dermatological compositions for delivering active agents to the skin are known to the art; for example, see U. S. Patent Nos. 4,992,478, 4,820,508, 4,608,392, and 4,559,157. Such dermatological compositions can be used in combinations with the compounds described herein where an ingredient of such compositions can optionally be replaced by a compound described herein, or a compound described herein can be added to the composition.

[0294] In some embodiments, depending on the type of a pharmaceutically acceptable carrier used, the compositions described herein can comprise, or alternatively consist essentially of, or yet further consist of about 0.1-100%, 0.1-50%, or 0.1-30%, such as 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,

[0295] 41

[0296] 530.045W01

[0297] 2024-123-02 75%, 80%, 85%, 90% or 95% of the pharmaceutically acceptable carrier used in the total weight of the composition, or any range between two of the numbers (end point inclusive).

[0298] In some embodiments, any one of the above listed pharmaceutically acceptable carriers is expressly excluded.

[0299] In some embodiments, the vesicles described herein are frozen (e.g., snap-frozen) or freeze-dried (e.g., lyophilized) to promote stability, preserve activity and increase shelf-life. One skilled in the art would understand how to reconstitute the lyophilized product before use.

[0300] In some embodiments, the populations of vesicles described herein are used immediately after isolation. In other embodiments, the populations of cell-derived vesicles are cryopreserved (e.g. frozen), for example, using any cryopreservation techniques well-known to those skilled in the art. In some embodiments, all or substantially of the cells and / or cellular debris are removed from the culture medium prior to cryopreservation. In some embodiments, all or substantially of the cells and / or cellular debris are removed from the culture medium after cryopreservation.

[0301] In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of a polyethylene glycol (PEG, e.g., PEG 150 Distearate), honey, a large molecular weight protein (e.g., bovine serum albumin or soy protein), polyvinyl alcohol, glyceryl monostearate, hyaluronic acid, glycerin, preferably vegetable-derived, proteins, preferably hydrolyzed proteins, (e.g., soy protein and silk protein), vasoline, citrosept, parabens, xanthan gum, i-carregaan, phytagel, Carbopol© polymers, and polyvinyl pyrrolidone.

[0302] In some embodiments, exosomes are preserved in serum albumin. Non-limiting examples of serum albumins appropriate for preservation of exosomes include bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin (OVA), and lactalbumin.

[0303] Useful dosages of the compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U. S. Patent No. 4,938,949 (Borch et al.). The amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.

[0304] In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg / kg, e.g., from about 10 to about 75 mg / kg of body weight per day, such as 3 to about 50

[0305] 42

[0306] 530.045W01

[0307] 2024-123-02 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg / kg / day, most preferably in the range of 15 to 60 mg / kg / day.

[0308] The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

[0309] The compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg / m2, conveniently 10 to 750 mg / m2, most conveniently, 50 to 500 mg / m2of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0310] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0311] The compounds described herein can be effective anti-tumor agents and have higher potency and / or reduced toxicity as compared to BHPI. Preferably, compounds of the invention are more potent and less toxic than BHPI, and / or avoid a potential site of catabolic metabolism encountered with BHPI, i.e., have a different metabolic profile than BHPI. Furthermore, the compounds described herein cause less severe ataxia than BHPI and other known compounds.

[0312] The invention provides therapeutic methods of treating cancer in a vertebrate such as a mammal, which involve administering to a mammal having cancer an effective amount of a compound or composition described herein. A mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like. Cancer refers to any of the various type of malignant neoplasm, which are in general characterized by an undesirable cellular proliferation, e.g., unregulated growth, lack of differentiation, local tissue invasion, and metastasis. Cancers that can be treated by a compound described herein include, for example, breast cancer, cervical carcinoma, colon cancer, endometrial cancer, leukemia, lung cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, or uterine cancer, and in particular, AML.

[0313] The ability of a compound of the invention to treat cancer may be determined by using assays well known to the art. For example, the design of treatment protocols, toxicity

[0314] 43

[0315] 530.045W01

[0316] 2024-123-02 evaluation, data analysis, quantification of tumor cell kills, and the biological significance of the use of transplantable tumor screens are known.

[0317] The vesicles and compositions herein can be administered to the subject by any method known by those of skill in the art. In some embodiments, the compositions are administered by I V. infusion, intravenous injection, direct injection, intramuscular injection, intracranial injection, or topically.

[0318] The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

[0319] EXAMPLES

[0320] Example 1. Materials and Methods

[0321] Materials. Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Corning Inc. (Corning, NY). Fetal bovine serum (FBS), Opti-modified Eagle’s medium (Opti-MEM), Pierce Coomassie Plus (Bradford) assay kits, QuantaBlu fluorogenic peroxidase substrate were purchased from Thermo Fisher Scientific (Waltham, MA). Expi293F expression medium was purchased form FUJIFILM Irvine Scientific Inc. (Santa Ana, CA). PELMax transfection reagent was purchased from Polysciences Inc. (Warrington, PA).

[0322] Cell culturing. U937, HL60, KG1A, HEK293T, and Jurkat cells were obtained from American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium supplemented with 10% FBS. HEK293T cells were cultured with DMEM medium with 10% FBS. Expi293F cells were purchased from Thermo Fisher Scientific (Waltham, MA) and cultured with Expi293F expression medium with 125 rpm at 37 °C and 5% CO2. Human peripheral blood mononuclear cells (PBMCs) were purchased from HemaCare (Van Nuys, CA).

[0323] Molecular cloning. Synthetic genes encoding aCLL-1 and aCD3 scFvs derived from aCLL-1 IgG (clone 1075.7) and aCD3 IgG (clone UCHT1) antibodies, respectively, were purchased from Integrated DNA Technologies, Inc. (Skokie, IL). Each scFv includes N-terminal light chain variable region (VL) and C-terminal heavy chain variable region (VH) separated by a flexible (GGGGS)s (SEQ ID NO: 52) linker. A gene fragment encoding aCD3-aCLL-1 dual-scFv with a flexible (GGGGS)s (SEQ ID NO: 52) linker between scFvs was generated through overlap extension polymerase chain reaction (PCR) using primers listed in Table 1. To generate mammalian expression constructs, single and dual-scFv gene fragments 44

[0324] 530.045W01

[0325] 2024-123-02 were subcloned into pDisplay vector (Thermo Fisher Scientific, MA) between Bglll and Sall restriction enzyme sites, resulting in the fusion proteins of HA-scFv antibody-platelet-derived growth factor receptor transmembrane domain (PDGFR TMD).

[0326] A PD-1-CD9 fusion fragment with an N-terminal HA tag was prepared through overlap extension PCR using PD-1 and CD9 DNA templates obtained from Horizon Discovery Ltd (Waterbeach, United Kingdom). Synthetic gene encoding human CD70 with a C-terminal His6tag was purchased from Integrated DNA Technologies, Inc. (Skokie, IL). A HA-PD-1-CD9-CD70-His6 fusion gene fragment was generated by overlap extension PCR. A GGGGS (SEQ ID NO: 12) linker was placed between PD-1 and CD9 fragment and a (GGGGS)₂ (SEQ ID NO: 53) linker was placed between CD9 and CD70. The HA-PD-l-CD9-CD70-His6 fusion gene fragment was subcloned into pDisplay vector between the Bglll and Notl restriction enzyme sites.

[0327] Expression and purification of exosomes. Expi293F cells were transiently transfected with the expression constructs confirmed by DNA sequencing using PEI MAX 40K transfection reagent. Cell culture supernatants containing exosomes were collected on day 3 and 6 after transfection.

[0328] Exosomes were isolated through differential centrifugation and ultracentrifugation at 4°C as our previous study with modifications.8Briefly, cell cultures were first centrifuged at 100×g for 10 min to remove Expi293F cells. Supernatants were collected and centrifuged at 4,000×g for 30 min and then 14,000×g for 1 h to remove dead cells and cell debris. Clarified supernatants were centrifuged at 256,000×g for 2 h with a Type 70 Ti rotor by Optima L-80 XP ultracentrifuge (Beckman Instruments, CA) to pellet exosomes. After washing twice with PBS, pellets were resuspended in PBS, followed by filtration with 0.22 pm filters. Protein concentrations of purified exosomes were determined by Bradford assay kits by following manufacturer’s instruction.

[0329] Immunoblot analysis. Briefly, 5 pg of exosome samples were boiled with 1 pL of 100 mM dithiothreitol (DTT) in 10 pL of NuPAGE LDS sample buffer (Thermo Fisher Scientific, MA) at 98°C for 10 min, separated in 4%-20% ExpressPlus-PAGE gels (GeneScript, Piscataway, NJ, USA), and then transferred to immune blot PVDF membranes (Bio-Rad Laboratories, CA, USA). After incubation with 5% BSA in PBST (PBS with 0.1% Tween-20) for 2 h at room temperature, membranes were incubated with appropriate diluted primary antibodies overnight at 4°C, which included anti-HA (2-2.2.14; Thermo Fisher Scientific, MA, USA), anti-CD9 (D8O1A; Cell Signaling Technology, MA, USA), anti-CD63 (H5C6; BioLegend, CA, USA), and anti-CD81 (1.3.3.22; Thermo Fisher Scientific, MA, USA).

[0330] 45

[0331] 530.045W01

[0332] 2024-123-02 Following incubation with secondary antibodies (Thermo Fisher Scientific, MA, USA), membranes were developed and imaged using a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, CA, USA) after additions of SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, MA, USA).

[0333] Nanoparticle tracking analysis. The size distribution and concentration of purified exosomes were determined through nanoparticle tracking analysis using a Nanosight LM10 (Malvern Instruments, UK) by following the manufacturer’s instruction. Six replicates of analysis with 60 s for each sample were performed.

[0334] ELISA analysis of binding of exosomes to ligands and receptors. To evaluate binding of PD-1-CD70 Exos to both PD-L1 and CD70, sandwich ELISA was performed with His6-CD27 as the capture protein and PD-Ll-hFc as the detection reagent. The binding of aCD3-aCLL-l-PD-l-CD70 PRIME-Exos to PD-L1, PD-L2, and CD27 were also detected by ELISA. High-binding 96-well plates (Greiner Bio-One, Monroe, NC, USA) were coated with exosomes at various concentrations overnight at room temperature. After extensively washing with PBST (PBS with 0.1% Tween-20), wells were blocked with 5% BSA in PBS for 4 h at room temperature and washed with PBST. Corresponding ligands or receptors (PD-Ll-hFc and PD-L2-hFc from PeproTech, Inc. and CD27-Hise from Sino Biological Inc.) were incubated for 2 h at room temperature, followed by 5 times extensive washes with PBST. Goat anti -human IgG HRP or mouse anti-Hise HRP was subsequently added for 1 h incubation at room temperature. QuantaBlu fluorogenic peroxidase substrate (Thermo Fisher Scientific) was then added after 5 times extensive washes with PBST. Fluorescence intensities (Ex: 325 nm; Em: 420 nm) were measured by a BioTek Synergy H1 Hybrid Multi-Mode Microplate reader (BioTek, VT).

[0335] Flow cytometry of antigen expression. To analyze cell-surface expression levels of CLL-1 and CD3, U937, HL-60, KG1A, and Jurkat cells were stained with APC anti-CLL-1 antibody (50C1, BioLegend, CA) or APC anti-CD3 antibody (UCHT1, BioLegend, CA) on ice. To measure cell-surface expression levels of PD-L1 and PD-L2, U937, HL60, KG1A, and HEK293T cells were first treated with human PBMCs (PBMCtarget cell = 2: 1) in the absence or presence of aCD3-aCLL-l-Exos (1000 ng mL’1) or IFN-y (100 U mL’1) for 48 h. Cells were then stained with PE anti-PD-Ll (clone: 29E.2A3, BioLegend) and APC anti-PD-L2 (clone: MIH18, BioLegend). After washing with PBS, cells were analyzed using a BD Fortessa X20 flow cytometer (BD Biosciences, CA). All flow cytometry data were processed by FlowJo_V10 software (Tree Star, USA).

[0336] 46

[0337] 530.045W01

[0338] 2024-123-02 Flow cytometry of binding of engineered exosomes to target cells. U937, HL60, KG1A, and Jurkat cells were incubated with exosomes (0.1 mg ml1) in PBS for 30 min on ice. After washing three times with PBS, cells were incubated with an anti -HA primary antibody (2-2.2.14; Thermo Fisher Scientific, MA, USA) for 30 min on ice. Following three washes with PBS, cells were incubated with an Alexa Fluor 488-conjugated anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific, MA, USA) for 30 min on ice. Samples were washed three times with PBS and analyzed using a BD Fortessa X20 flow cytometer. All flow cytometry data were processed with FlowJo_V10 software (Tree Star, OR, USA).

[0339] In vitro cytotoxicity. Cytotoxicity of exosomes were evaluated using non-activated human PBMC as effector cells and carboxyfluorescein succinimidyl ester (CFSE) (BioLegend, CA, USA)-labeled AML or HEK293T cells as targeted cells. Target cells (1 x 105per well) were mixed with PBMCs (8×105per well) and incubated with various concentrations of exosomes in 24-well plates for 24 h. The viability of cells was analyzed by flow cytometry.

[0340] T cell activation analysis. T cell activation assays were performed as previously described in Cheng etal., J Am ChemSoc 140, 16413-16417 (2018), with modifications Briefly, non-activated hPBMCs (5×104per well) were incubated with target cells (2.5×104per well) in the presence of different types of exosomes (1000 ng mL1) in 96-well cell culture plates for 24, 48, 72, or 96 h. Cells were collected and stained with an Alexa Fluor 488 anti-CD3 (UCHT1, BioLegend, CA), PE anti-CD25 (M-A251, BioLegend, CA), APC anti-CD69 (FN50, BioLegend, CA), followed by flow cytometric analysis using a BD Fortessa X20 flow cytometer (BD Biosciences, CA). The levels of secreted IFN-y and IL-2 in collected culture media were measured using ELISA kits (R& D System, MN).

[0341] In vivo efficacy study. Six-week-old female

[0342]

[0343] , NOD.Cg- Il2rmlWjlI zi (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA).

[0344] Luciferase-expressing U937 cells (one million per mouse) were inoculated by tail vein injection. Meanwhile, human PBMC (2×106cells mL'1) were cultured in RPMI 1640 medium with 10% FBS and stimulated with immobilized anti-human CD3 antibody (clone: OKT3, BioLegend), soluble anti-CD28 antibody (clone: 28.2, BioLegend), and recombinant human interleukin-2 (IL-2) (40 IU mL'1, BioLegend) in flasks for 3 days. Cells were then expanded with human IL-2 (40 IU mL'1) in RPMI 1640 medium plus 10% FBS. Mice were randomized into six groups and received two intraperitoneal injections of 2×107expanded human PBMCs on day 1 and 7 post U937 cells inoculation, followed by intravenous treatment with PBS (vehicle only), native exosomes (10 mg kg'1), PD-1-CD70 Exos (10 mg kg'1), aCD3-aCLL-l Exos (10 mg kg'1), a combination of aCD3-aCLL-l Exos (10 mg kg'1) and PD-1-OX40L Exos

[0345] 47

[0346] 530.045W01

[0347] 2024-123-02 (10 mg kg-1), or aCD3-aCLL-l-PD-l-CD70 PRIME Exos (10 mg kg1) on day 2, 4, 6, 8, 10, and 12 postU937 cell inoculation. Following intraperitoneal injection of D-luciferin (150 mg kg'1) (Syd Labs, MA, USA), mice were imaged for luminescence signals using an in vivo imaging system (IVIS) Lumina III imaging system (PerkinElmer, MA, USA) on day 3, 10, 13, and 15 after U937 cell inoculation. At the end of the study, mice were euthanized, and major organs and tissues were collected for weighing and / or analysis.

[0348] In vivo safety study. Human PBMC (2×106cells mL’1) were first cultured and expanded as described above. Six-week-old female NSG mice purchased from the Jackson Laboratory (Bar Harbor, ME, USA) were randomized into PBS and aCD3-aCLL-l-PD-l-CD70 PRIME Exos groups. Mice received two intraperitoneal injections of 2×107expanded human PBMCs on day 0 and 6 and were intravenously administered with PBS or aCD3-aCLL-1-PD-1-CD70 PRIME Exos (10 mg kg1) on day 1, 3, 5, 7, 9, and 11 post the first PBMC injection. At the end of the study, mice were euthanized, and major organs and tissues were collected for weighing and / or analysis.

[0349] Lymphocyte isolation and analysis. Blood, spleen, and bone marrow samples from all groups were prepared for single-cell suspensions. Cells were treated with red blood cell lysis buffer (BioLegend, CA, USA) by following the manufacturer’s instruction. Thereafter, cells were stained for live and dead cells with live / dead-fixable Zombie Aqua (BioLegend, CA, USA). After washing three times, samples were performed with one of following three cell surface marker staining options (1) PE anti-mouse CD34 antibody (119308, Biolegend, CA, USA), Pacific Blue anti-mouse CD45 antibody (MCD4528, Thermo Fisher Scientific, MA, USA), and APC anti-human CLL-1 antibody (353606, Biolegend, CA, USA); (2) PerCP / Cyanine5.5 anti-human CD45 antibody (clone: 2D1, BioLegend, CA, USA), FITC antihuman CD3 antibody (clone: UCHT1, BioLegend, CA, USA), APC / Cyanine7 anti-human CD4 antibody (clone: OKT4, BioLegend, CA, USA), Pacific blue anti-human CD8 antibody (clone: RPA-T8, BioLegend, CA, USA), PE anti-human CD25 antibody (clone: M-A251, BioLegend, CA, USA), and PE / Dazzle 594 anti-human CD127 antibody (clone: A019D5, BioLegend, CA, USA); (3) PerCP / Cyanine5.5 anti-human CD45 antibody (clone: 2D1, BioLegend, CA, USA), FITC anti-human CD3 antibody (clone: UCHT1, BioLegend, CA, USA), APC / Cyanine7 antihuman CD4 antibody (clone: OKT4, BioLegend, CA, USA), Pacific blue anti-human CD8 antibody (clone: RPA-T8, BioLegend, CA, USA), PE anti -human CD25 antibody (clone: M-A251, BioLegend, CA, USA), and PE / Dazzle 594 anti -human FoxP3 antibody (clone: A019D5, BioLegend, CA, USA). To stain intracellular FoxP3 with PE / Dazzle 594 anti-human FoxP3 antibody (clone: 206D, BioLegend, CA, USA), cells were fixed and permeabilized with an

[0350] 48

[0351] 530.045W01

[0352] 2024-123-02 eBioscience FOXP3 / transcri ption factor staining buffer set kit (Thermo Fisher Scientific, MA, USA) by following the manufacturer’s instruction.

[0353] All samples were analyzed by a BD Fortessa X-20 Cell Analyzer (CA, USA) and data were processed with FlowJo software (Tree Star, OR, USA). Total numbers of CD4+, CD8+, CD4+CD25+CD127‘, and CD4+CD25+FoxP3+cells were analyzed within CD45+hematopoietic cell populations using FlowJo_V10 software (Tree Star, OR, USA).

[0354] Data for all samples were first gated based on morphology (FSC-A / SSC-A) to remove non-cell debris, and then Zombie Aqua-staining negative (live cells) populations were analyzed on fluorescence intensities of different antibodies used for staining.

[0355] Creatinine colorimetric assay. To measure concentrations of creatinine in plasma, plasma samples collected at the end of animal study were mixed with equal volume of 1.2 M trichloroacetic acid (Oakwood Chemicals, SC, USA). After centrifugation at 12,000 ×g for 20 min, supernatants were collected and incubated with twice the volume of working solution (38 mM picric acid premixed with equal volume of 1.2 M NaOH). After incubation at room temperature for 20 min, absorbance at 510 nm were recorded by a Synergy H1 plate reader (BioTek, VT, USA). Creatinine standard solutions in a series of concentrations were utilized in the assay for extrapolating a standard curve.

[0356] ALT activity assay. To determine alanine aminotransferase (ALT) activity in plasma, plasma samples (5 pL) collected at the end of animal study and standard sodium pyruvate solution were first incubated with 25 pL of substrate solution (0.2 M alanine and 2 mM 2-oxoglutarate in 0.1 M disodium phosphate, pH 7.4) for 1 h at 37°C in 96-well plates. Then, the mixture was incubated with 25 pL of 1 mM 2,4-dinitrophenylhydrazine in 1 M HC1 for 20 min at room temperature. After incubation with 250 pL of 0.5 M NaOH at room temperature. The absorbance at 510 nm of mixture was measured using a Synergy H1 plate reader (BioTek, VT, USA). A series of pyruvate standard solutions were prepared for extrapolating a standard curve.

[0357] Statistical analysis. All statistical analyses were performed using GraphPad Prism (GraphPad Software, CA, USA). The statistical analyses were performed on independent biological replicates, n >2 for in vitro assays and n = 5 for in vivo studies. Statistical significance between two groups was determined using two-tailed student t tests. One-way ANOVA analyses were performed for comparisons of multiple groups. Significance of finding was defined as follows: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. All data are shown as mean ± SD.

[0358] Table 1. List of primers used for molecular cloning.

[0359] 49

[0360] 530.045W01

[0361] 2024-123-02 Name Sequence

[0362] aCD3 5'GGCCAGATCTGATATCCAGATGACACAGACAACCTCAAGTCTTAG- scFv 3' (SEQ ID NO: 37)

[0363] Forward

[0364] aCD3 5'-CCTGAGCCTCCCCCGCCTGATCCGCCACCGCCGC

[0365] scFv TGCTAACGGTAACGGTGGTACC-3' (SEQ ID NO: 38)

[0366] Reverse

[0367] aCLL-1 5'-AGGCGGGGGAGGCTCAGGCGGAGGTGGCAGCG

[0368] scFv AGAACGTGCTCACCCAATCCCC-3' (SEQ ID NO: 39)

[0369] Forward

[0370] aCLL-1 5'-GTTCGTCGACGCTTCCGCCACCCCCAGACACGG

[0371] scFv TCACGCTGGTGC-3' (SEQ ID NO: 40)

[0372] Reverse

[0373] aCD3- 5'-AGGCGGGGGAGGCTCAGGCGGAGGTGGCAGCG

[0374] aCLL-1 AGAACGTGCTCACCCAATCCCC-3' (SEQ ID NO: 41)

[0375] scFv

[0376] overlap

[0377] Forward

[0378] aCD3- 5'-CCTGAGCCTCCCCCGCCTGATCCGCCACCGCCGC

[0379] aCLL-1 TGCTAACGGTAACGGTGGTACC-3' (SEQ ID NO: 42)

[0380] scFv

[0381] overlap

[0382] Reverse

[0383] PD-1- 5'-CCGGCCAGATCTTTCTTAGACTCCCCAGACAGGCCCT-3' (SEQ ID CD9 NO: 43)

[0384] Forward

[0385] PD-1- 5 '-CCC ACCTCC ACCGCTACC ACCGCCTCCGACC ATCTCGCGGT-3 ' CD9 (SEQ ID NO: 44)

[0386] Reverse

[0387] CD70 5'-TGGGTCCGTCGACCCGGAGGAGGGTTCGGGCTGCT-3' (SEQ ID Forward NO: 45)

[0388] CD70 5'GATCTCGAGCGGCCGCCTTAATGGTGGTGGTGATGGTGGGG

[0389]

[0390] Reverse GCGCACCCACTGCACTCCA-3' (SEQ ID NO: 46)

[0391] Example 2. Amino Acid Sequences

[0392] 1.) First fusion protein: HA tag-anti-CD3-Li-anti-CLL-l-L2-Myc tag-TMD YPYDVPDYAGAQPARSDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPD GTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFA GGTKLEIKGGGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYT MNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTS EDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSENVLT QSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGGGGSGGGGSGG GGSDIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISY DGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAM DYWGQGTSVTVSGGGGSVDEQKLISEEDLNAVGQDTQEVIVVPHSLPFKVVVISAIL ALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 1)

[0393] 2.) Second fusion protein: HA tag-PD-1 protein-L3-TMD-L4-CD70 protein-6x His tag 50

[0394] 530.045W01

[0395] 2024-123-02 YPYDVPDYAGAQPARSFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVL NWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT YLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLL GSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLASGGG GSPVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYT GVYILIGAGALMMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKD EVIKEVQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDV LETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMVGGG GSGGGGSVDPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLP LESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYM VHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGD TLCTNLTGTLLPSRNTDETFFGVQWVRPHHHHHH (SEQ ID NO: 2)

[0396] 3.) Anti-CD3 scFv. CDRs are bolded / italicized.

[0397] DIQMTQTTSSLSASLGDRVTISCT NgDZRNFZAWYQQKPDGTVKLLIYFZSRZZ / SGVPSKFS GSGSGTDYSLTISNLEQEDIATYFCOOGW / ZPRTFAGGTKLEIKGGGSGGGSGGGSGGGSEV QLQQSGPELVI< PGASMI< ISCI< ASGYSFTGF77V7MVVI< QSHGI< NLEWMG / - / NPFA'GKS’7’F > A' FADKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGFFGDA'DPPFFDFWGAGTTVTVSS (SEQ ID NO: 3)

[0398] 4.) Anti-CLL-1 scFv. CDRs are bolded / italicized.

[0399] ENVLTQSPAIMSASPGEI< VTMTC / ? lASWr / AS’Fr / 7WYQQRSGASPI< LWIYA71SW7AAGVPARF SGSGSGTSYSLTISSVEAEDAATYYCOOES’G’F / 7FGAGTI< LELGGGGSGGGGSGGGGSDIQL QESGPGLVKPSQSLSLTCSVTGYSITA4FFW VWIRQFPGNKLEWMGFZSFDGFAWEVP5ZAAR ISITRDTSKNQFFLKLNSVTTEDTATYYC AKEGDFDrGWFFlA / DFWGQGTSVTVS (SEQ ID NO: 4)

[0400] 5.) PD1 Protein (UniprotNo. Q15116):

[0401] FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLA AFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKES LRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSR AARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATI VFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 5)

[0402] 6.) CD70 Protein (UniprotNo. P32970):

[0403] PEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVA ELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAIC SSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTL LPSRNTDETFFGVQWVRP (SEQ ID NO: 6)

[0404] 7.) PDGFR TMD (UniprotNo. P09619):

[0405] AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 7)

[0406] 8.) CD9 TMD (UniprotNo. P21926):

[0407] PVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYTG VYILIGAGALMMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKDE VIKEVQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVL

[0408] 51

[0409] 530.045W01

[0410] 2024-123-02 ETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMV (SEQ ID NO: 8)

[0411] 9.) Hemagglutinin (HA) Tag:

[0412] YPYDVPDYA (SEQ ID NO: 9)

[0413] 10.) Myc Tag:

[0414] EQKLISEEDL (SEQ ID NO: 10)

[0415] 11.) 6X Histidine Tag:

[0416] HHHHHH (SEQ ID NO: 11)

[0417] 12.) Linker

[0418] GGGGS (SEQ ID NO: 12)

[0419] 13.) First fusion protein: anti-CDS-Li-anti-CLL-l-Li TMD GAQPARSDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYY TSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKG GGSGGGSGGGSGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQS HGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSAS PGEKVTMTCRAS SNVIS S YVHWYQQRSGASPKLWIYSTSNL ASGVPARF SGSGSGTS YSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGGGGSGGGGSGGGGSDIQLQE SGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPS LKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSV TVSGGGGSVDNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKP

[0420] R (SEQ ID NO: 13)

[0421] 14.) Second fusion protein: PD-1 protein-L3-TMD-L4-CD70 protein GAQPARSFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSN QTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAP KAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWV LAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQ TEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLASGGGGSPVKGGTK CIKYLLFGFNFIFWL AGIAVLAIGLWLRFD SQTKSIFEQETNNNNS SF YTGVYILIGAG ALMMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKDEVIKEVQEF YKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKSC PDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMVGGGGSGGGGSV DPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDV AELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAI CSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGT LLPSRNTDETFFGVQWVRP (SEQ ID NO: 14)

[0422] 15. DNA sequence encoding the fusion protein of SEQ ID NO: 1

[0423] 52

[0424] 530.045W01

[0425] 2024-123-02 tatccatatgatgttccagattatgctggggcccagccggccagatctgatatccagatgacacagacaacctcaagtcttagtgcatca ctgggagatcgtgtgactataagctgccgcgcatcacaggacattcgcaattatctgaattggtatcaacagaagcctgatggcaccgt gaaacttctgatctattacaccagtcgtctgcatagcggtgttccgagcaaattttcaggctcagggtcaggaaccgattattcactgacg attagtaatttagaacaagaagatattgcaacctatttctgtcaacagggtaataccctgccgtggacctttgcaggtggtaccaaactgg aaattaaaggaggtggcagtggagggggaagcggcggcggttcaggaggcggttctgaggtccagttacagcagagcggtccgg aactggttaaaccgggtgcaagcatgaaaattagctgtaaagcaagcggttatagctttaccggttataccatgaattgggttaaacaga gccatggtaaaaatctggaatggatgggtctgattaatccgtataaaggtgttagcacctataatcagaaatttaaagataaagcaaccct gaccgttgataaaagcagcagcaccgcatatatggaactgctgagcctgaccagcgaagatagcgccgtttactattgcgcacgcag cggttattatggtgatagcgattggtattttgatgtttggggtgcaggtaccaccgttaccgttagcagcggcggtggcggatcaggcgg gggaggctcaggcggaggtggcagcgagaacgtgctcacccaatcccccgccattatgtccgcctccccaggcgaaaaggtgaca atgacctgcagggccagctccaacgtgatcagctcttacgtgcactggtaccagcaacggtccggcgcctcccctaagctgtggatct atagcacaagcaacctggcttccggcgtgcctgcacggttcagcggaagcggaagcggaacaagttactccctcaccatttctagcgt tgaagccgaggatgccgctacatactattgtcaacagtacagcggataccccctgaccttcggagccggcacaaaactggagctcgg cgggggcggatcaggaggcgggggtagcggcgggggaggctccgacatccagctgcaggagagcggccccggcctggtgaag cccagccagagcctgagcctgacctgcagcgtgaccggctacagcatcaccagcgcctattactggaactggatccggcagttcccc ggcaacaagctggagtggatgggctacatcagctacgacggccggaacaactacaacccaagcctgaagaaccggatcagcatca cccgggacaccagcaagaaccagtttttcctgaagctgaacagcgtgaccacagaggacaccgccacctattactgcgccaaggag ggagactacgacgtgggcaactactacgccatggactactggggccagggcaccagcgtgaccgtgtctgggggtggcggaagc gtcgacgaacaaaaactcatctcagaagaggatctgaatgctgtgggccaggacacgcaggaggtcatcgtggtgccacactccttg ccctttaaggtggtggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcatcctcatcatgctttggcagaagaa gccacgt (SEQ ID NO: 15)

[0426] 16. DNA sequence encoding the fusion protein of SEQ ID NO: 2 tatccatatgatgttccagattatgctggggcccagccggccagatctttcttagactccccagacaggccctggaacccccccaccttc tccccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcagcttctccaacacatcggagagcttcgtgctaaact ggtaccgcatgagccccagcaaccagacggacaagctggccgctttccccgaggaccgcagccagcccggccaggactgccgct tccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgtg gggccatctccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtg cccacagcccaccccagcccctcacccaggccagccggccagttccaaaccctggtggttggtgtcgtgggcggcctgctgggca gcctggtgctgctagtctgggtcctggccgtcatctgctcccgggccgcacgagggacaataggagccaggcgcaccggccagcc cctgaaggaggacccctcagccgtgcctgtgttctctgtggactatggggagctggatttccagtggcgagagaagaccccggagcc ccccgtgccctgtgtccctgagcagacggagtatgccaccattgtctttcctagcggaatgggcacctcatcccccgcccgcaggggc tcagccgacggccctcggagtgcccagccactgaggcctgaggatggacactgctcttggcccctcgctagcggaggcggtggtag tccggtcaaaggaggcaccaagtgcatcaaatacctgctgttcggatttaacttcatcttctggcttgccgggattgctgtccttgccattg gactatggctccgattcgactctcagaccaagagcatcttcgagcaagaaactaataataataattccagcttctacacaggagtctatat tctgatcggagccggcgccctcatgatgctggtgggcttcctgggctgctgcggggctgtgcaggagtcccagtgcatgctgggact gttcttcggcttcctcttggtgatattcgccattgaaatagctgcggccatctggggatattcccacaaggatgaggtgattaaggaagtc caggagttttacaaggacacctacaacaagctgaaaaccaaggatgagccccagcgggaaacgctgaaagccatccactatgcgtt gaactgctgtggtttggctgggggcgtggaacagtttatctcagacatctgccccaagaaggacgtactcgaaaccttcaccgtgaagt cctgtcctgatgccatcaaagaggtcttcgacaataaattccacatcatcggcgcagtgggcatcggcattgccgtggtcatgatatttg gcatgatcttcagtatgatcttgtgctgtgctatccgcaggaaccgcgagatggtcggaggcggtggtagcggtggaggtgggtccgt cgacccggaggagggttcgggctgctcggtgcggcgcaggccctatgggtgcgtcctgcgggctgctttggtcccattggtcgcgg gcttggtgatctgcctcgtggtgtgcatccagcgcttcgcacaggctcagcagcagctgccgctcgagtcacttgggtgggacgtagc tgagctgcagctgaatcacacaggacctcagcaggaccccaggctatactggcaggggggcccagcactgggccgctccttcctgc atggaccagagctggacaaggggcagctacgtatccatcgtgatggcatctacatggtacacatccaggtgacgctggccatctgttc ctccacgacggcctccaggcaccaccccaccaccctggccgtgggaatctgctctcccgcctcccgtagcatcagcctgctgcgtct cagcttccaccaaggttgtaccattgcctcccagcgcctgacgcccctggcccgaggggacacactctgcaccaacctcactgggac acttttgccttcccgaaacactgatgagaccttctttggagtgcagtgggtgcgcccccaccatcaccaccaccat (SEQ ID NO: 16)

[0427] 53

[0428] 530.045W01

[0429] 2024-123-02 17. DNA sequence encoding the fusion protein of SEQ ID NO: 13 ggggcccagccggccagatctgatatccagatgacacagacaacctcaagtcttagtgcatcactgggagatcgtgtgactataagct gccgcgcatcacaggacattcgcaattatctgaattggtatcaacagaagcctgatggcaccgtgaaacttctgatctattacaccagtc gtctgcatagcggtgttccgagcaaattttcaggctcagggtcaggaaccgattattcactgacgattagtaatttagaacaagaagatat tgcaacctatttctgtcaacagggtaataccctgccgtggacctttgcaggtggtaccaaactggaaattaaaggaggtggcagtggag ggggaagcggcggcggttcaggaggcggttctgaggtccagttacagcagagcggtccggaactggttaaaccgggtgcaagcat gaaaattagctgtaaagcaagcggttatagctttaccggttataccatgaattgggttaaacagagccatggtaaaaatctggaatggat gggtctgattaatccgtataaaggtgttagcacctataatcagaaatttaaagataaagcaaccctgaccgttgataaaagcagcagcac cgcatatatggaactgctgagcctgaccagcgaagatagcgccgtttactattgcgcacgcagcggttattatggtgatagcgattggta ttttgatgtttggggtgcaggtaccaccgttaccgttagcagcggcggtggcggatcaggcgggggaggctcaggcggaggtggca gcgagaacgtgctcacccaatcccccgccattatgtccgcctccccaggcgaaaaggtgacaatgacctgcagggccagctccaac gtgatcagctcttacgtgcactggtaccagcaacggtccggcgcctcccctaagctgtggatctatagcacaagcaacctggcttccg gcgtgcctgcacggttcagcggaagcggaagcggaacaagttactccctcaccatttctagcgttgaagccgaggatgccgctacat actattgtcaacagtacagcggataccccctgaccttcggagccggcacaaaactggagctcggcgggggcggatcaggaggcgg gggtagcggcgggggaggctccgacatccagctgcaggagagcggccccggcctggtgaagcccagccagagcctgagcctga cctgcagcgtgaccggctacagcatcaccagcgcctattactggaactggatccggcagttccccggcaacaagctggagtggatgg gctacatcagctacgacggccggaacaactacaacccaagcctgaagaaccggatcagcatcacccgggacaccagcaagaacca gtttttcctgaagctgaacagcgtgaccacagaggacaccgccacctattactgcgccaaggagggagactacgacgtgggcaacta ctacgccatggactactggggccagggcaccagcgtgaccgtgtctgggggtggcggaagcgtcgacaatgctgtgggccaggac acgcaggaggtcatcgtggtgccacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggtgctcaccatcatc tcccttatcatcctcatcatgctttggcagaagaagccacgt (SEQ ID NO: 17)

[0430] 18. DNA sequence encoding the fusion protein of SEQ ID NO: 14 ggggcccagccggccagatctttcttagactccccagacaggccctggaacccccccaccttctccccagccctgctcgtggtgacc gaaggggacaacgccaccttcacctgcagcttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaac cagacggacaagctggccgctttccccgaggaccgcagccagcccggccaggactgccgcttccgtgtcacacaactgcccaacg ggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgtggggccatctccctggcccccaag gcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcccctc acccaggccagccggccagttccaaaccctggtggttggtgtcgtgggcggcctgctgggcagcctggtgctgctagtctgggtcct ggccgtcatctgctcccgggccgcacgagggacaataggagccaggcgcaccggccagcccctgaaggaggacccctcagccgt gcctgtgttctctgtggactatggggagctggatttccagtggcgagagaagaccccggagccccccgtgccctgtgtccctgagcag acggagtatgccaccattgtctttcctagcggaatgggcacctcatcccccgcccgcaggggctcagccgacggccctcggagtgcc cagccactgaggcctgaggatggacactgctcttggcccctcgctagcggaggcggtggtagtccggtcaaaggaggcaccaagtg catcaaatacctgctgttcggatttaacttcatcttctggcttgccgggattgctgtccttgccattggactatggctccgattcgactctcag accaagagcatcttcgagcaagaaactaataataataattccagcttctacacaggagtctatattctgatcggagccggcgccctcatg atgctggtgggcttcctgggctgctgcggggctgtgcaggagtcccagtgcatgctgggactgttcttcggcttcctcttggtgatattcg ccattgaaatagctgcggccatctggggatattcccacaaggatgaggtgattaaggaagtccaggagttttacaaggacacctacaa caagctgaaaaccaaggatgagccccagcgggaaacgctgaaagccatccactatgcgttgaactgctgtggtttggctgggggcgt ggaacagtttatctcagacatctgccccaagaaggacgtactcgaaaccttcaccgtgaagtcctgtcctgatgccatcaaagaggtctt cgacaataaattccacatcatcggcgcagtgggcatcggcattgccgtggtcatgatatttggcatgatcttcagtatgatcttgtgctgtg ctatccgcaggaaccgcgagatggtcggaggcggtggtagcggtggaggtgggtccgtcgacccggaggagggttcgggctgctc ggtgcggcgcaggccctatgggtgcgtcctgcgggctgctttggtcccattggtcgcgggcttggtgatctgcctcgtggtgtgcatcc agcgcttcgcacaggctcagcagcagctgccgctcgagtcacttgggtgggacgtagctgagctgcagctgaatcacacaggacct cagcaggaccccaggctatactggcaggggggcccagcactgggccgctccttcctgcatggaccagagctggacaaggggcag ctacgtatccatcgtgatggcatctacatggtacacatccaggtgacgctggccatctgttcctccacgacggcctccaggcaccaccc caccaccctggccgtgggaatctgctctcccgcctcccgtagcatcagcctgctgcgtctcagcttccaccaaggttgtaccattgcctc ccagcgcctgacgcccctggcccgaggggacacactctgcaccaacctcactgggacacttttgccttcccgaaacactgatgagac cttctttggagtgcagtgggtgcgcccc (SEQ ID NO: 18)

[0431] 54

[0432] 530.045W01

[0433] 2024-123-02 19. DNA sequence encoding the anti-CD-3 ScFv of SEQ ID NO: 3 Gatatccagatgacacagacaacctcaagtcttagtgcatcactgggagatcgtgtgactataagctgccgcgcatcacaggacattc gcaattatctgaattggtatcaacagaagcctgatggcaccgtgaaacttctgatctattacaccagtcgtctgcatagcggtgttccgag caaattttcaggctcagggtcaggaaccgattattcactgacgattagtaatttagaacaagaagatattgcaacctatttctgtcaacagg gtaataccctgccgtggacctttgcaggtggtaccaaactggaaattaaaggaggtggcagtggagggggaagcggcggcggttca ggaggcggttctgaggtccagttacagcagagcggtccggaactggttaaaccgggtgcaagcatgaaaattagctgtaaagcaagc ggttatagctttaccggttataccatgaattgggttaaacagagccatggtaaaaatctggaatggatgggtctgattaatccgtataaag gtgttagcacctataatcagaaatttaaagataaagcaaccctgaccgttgataaaagcagcagcaccgcatatatggaactgctgagc ctgaccagcgaagatagcgccgtttactattgcgcacgcagcggttattatggtgatagcgattggtattttgatgtttggggtgcaggta ccaccgttaccgttagcagc (SEQ ID NO: 19)

[0434] 20. DNA sequence encoding the anti-CLL-1 ScFv of SEQ ID NO: 4 Gagaacgtgctcacccaatcccccgccattatgtccgcctccccaggcgaaaaggtgacaatgacctgcagggccagctccaacgt gatcagctcttacgtgcactggtaccagcaacggtccggcgcctcccctaagctgtggatctatagcacaagcaacctggcttccggc gtgcctgcacggttcagcggaagcggaagcggaacaagttactccctcaccatttctagcgttgaagccgaggatgccgctacatact attgtcaacagtacagcggataccccctgaccttcggagccggcacaaaactggagctcggcgggggcggatcaggaggcgggg gtagcggcgggggaggctccgacatccagctgcaggagagcggccccggcctggtgaagcccagccagagcctgagcctgacct gcagcgtgaccggctacagcatcaccagcgcctattactggaactggatccggcagttccccggcaacaagctggagtggatgggc tacatcagctacgacggccggaacaactacaacccaagcctgaagaaccggatcagcatcacccgggacaccagcaagaaccagt ttttcctgaagctgaacagcgtgaccacagaggacaccgccacctattactgcgccaaggagggagactacgacgtgggcaactact acgccatggactactggggccagggcaccagcgtgaccgtgtct (SEQ ID NO: 20)

[0435] 21. DNA sequence encoding the PDGFR TMD of SEQ ID NO: 7 Gctgtgggccaggacacgcaggaggtcatcgtggtgccacactccttgccctttaaggtggtggtgatctcagccatcctggccctgg tggtgctcaccatcatctcccttatcatcctcatcatgctttggcagaagaagccacgt (SEQ ID NO: 21)

[0436] 22. DNA sequence encoding the PD-1 protein of SEQ ID NO: 5 Ttcttagactccccagacaggccctggaacccccccaccttctccccagccctgctcgtggtgaccgaaggggacaacgccaccttc acctgcagcttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaaccagacggacaagctggccgct ttccccgaggaccgcagccagcccggccaggactgccgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgt ggtcagggcccggcgcaatgacagcggcacctacctctgtggggccatctccctggcccccaaggcgcagatcaaagagagcctg cgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcccctcacccaggccagccggccagtt ccaaaccctggtggttggtgtcgtgggcggcctgctgggcagcctggtgctgctagtctgggtcctggccgtcatctgctcccgggcc gcacgagggacaataggagccaggcgcaccggccagcccctgaaggaggacccctcagccgtgcctgtgttctctgtggactatg gggagctggatttccagtggcgagagaagaccccggagccccccgtgccctgtgtccctgagcagacggagtatgccaccattgtct ttcctagcggaatgggcacctcatcccccgcccgcaggggctcagccgacggccctcggagtgcccagccactgaggcctgagga tggacactgctcttggcccctc (SEQ ID NO: 22)

[0437] 23. DNA sequence encoding the CD9 protein of SEQ ID NO: 8 Ccggtcaaaggaggcaccaagtgcatcaaatacctgctgttcggatttaacttcatcttctggcttgccgggattgctgtccttgccattg gactatggctccgattcgactctcagaccaagagcatcttcgagcaagaaactaataataataattccagcttctacacaggagtctatat tctgatcggagccggcgccctcatgatgctggtgggcttcctgggctgctgcggggctgtgcaggagtcccagtgcatgctgggact gttcttcggcttcctcttggtgatattcgccattgaaatagctgcggccatctggggatattcccacaaggatgaggtgattaaggaagtc caggagttttacaaggacacctacaacaagctgaaaaccaaggatgagccccagcgggaaacgctgaaagccatccactatgcgtt gaactgctgtggtttggctgggggcgtggaacagtttatctcagacatctgccccaagaaggacgtactcgaaaccttcaccgtgaagt cctgtcctgatgccatcaaagaggtcttcgacaataaattccacatcatcggcgcagtgggcatcggcattgccgtggtcatgatatttg gcatgatcttcagtatgatcttgtgctgtgctatccgcaggaaccgcgagatggtc (SEQ ID NO: 23)

[0438] 55

[0439] 530.045W01

[0440] 2024-123-02 24. DNA sequence encoding the CD70 protein of SEQ ID NO: 6 Ccggaggagggttcgggctgctcggtgcggcgcaggccctatgggtgcgtcctgcgggctgctttggtcccattggtcgcgggctt ggtgatctgcctcgtggtgtgcatccagcgcttcgcacaggctcagcagcagctgccgctcgagtcacttgggtgggacgtagctga gctgcagctgaatcacacaggacctcagcaggaccccaggctatactggcaggggggcccagcactgggccgctccttcctgcatg gaccagagctggacaaggggcagctacgtatccatcgtgatggcatctacatggtacacatccaggtgacgctggccatctgttcctc cacgacggcctccaggcaccaccccaccaccctggccgtgggaatctgctctcccgcctcccgtagcatcagcctgctgcgtctcag cttccaccaaggttgtaccattgcctcccagcgcctgacgcccctggcccgaggggacacactctgcaccaacctcactgggacactt ttgccttcccgaaacactgatgagaccttctttggagtgcagtgggtgcgcccc (SEQ ID NO: 24)

[0441] All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0442] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

[0443] 56

[0444] 530.045W01

[0445] 2024-123-02

Claims

What is claimed is:

1. An engineered extracellular vesicle comprising:a first fusion protein comprising a formula A-B-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain; and a second fusion protein comprising a formula D-E-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein;wherein both the first fusion protein and the second fusion protein are displayed on a surface of the engineered extracellular vesicle.

2. The engineered extracellular vesicle of claim 1, wherein each of the first antibody moiety and the second antibody moiety comprise a single chain variable fragment (scFv), a single domain antibody, a bispecific antibody, or a multispecific antibody.

3. The engineered extracellular vesicle of claim 1 or 2, wherein each of the first antibody moiety and the second antibody moiety comprise a single chain variable fragment (scFv).

4. The engineered extracellular vesicle of any one of claims 1-3, wherein the first transmembrane domain comprises a transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR) protein.

5. The engineered extracellular vesicle of any one of claims 1-4, wherein the second transmembrane domain comprises a transmembrane domain of a CD9 protein.

6. The engineered extracellular vesicle of any one of claims 1-5, wherein the first fusion protein and the second fusion protein are displayed on the surface of the engineered extracellular vesicle in a 1:1 ratio.

7. The engineered extracellular vesicle of any one of claims 1-6, wherein the extracellular vesicle comprises one or more of an exosome, a liposome, a microvesicle, an apoptotic body, and a combination thereof.

8. The engineered extracellular vesicle of any one of claims 1-7, wherein A is an N-terminus and C is the C-terminus of the first fusion protein.57530.045W012024-123-029. The engineered extracellular vesicle of any one of claims 1-8, wherein D is the N-terminus and F is the C-terminus of the second fusion protein10. The engineered extracellular vesicle of any one of claims 1-9, wherein the engineered extracellular vesicle comprises a particle size of about 25 nm to about 250 nm.

11. The engineered extracellular vesicle of any one of claims 1-10, wherein the first fusion protein comprises the formula A-L1-B-L2-C; andthe second fusion protein comprising the formula D-L3-E-L4-F;wherein L1 – L4 are linker sequences.

12. The engineered extracellular vesicle of claim 11, wherein the linker sequences L1 – L4 comprise the amino acid sequence (GGGGS)n (SEQ ID NO: 50), wherein n is 1, 2, 3, 4, or 5.

13. The engineered extracellular vesicle of claim 12, wherein at least one of the linker sequences Li - L4 comprises (GGGGS) (SEQ ID NO: 12), (GGGGS)₂ (SEQ ID NO: 53), or (GGGGS)3(SEQ ID NO: 52).

14. The engineered extracellular vesicle of any one of claims 1-13, wherein the first fusion protein and the second fusion protein further comprise one or more of a hemagglutinin epitope tag, a myc tag, and a 6x histidine epitope tag.

15. The engineered extracellular vesicle of any one of claims 1-14, wherein the first fusion protein comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 1 or SEQ ID NO: 13; andthe second fusion protein comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 2 or SEQ ID NO: 14.

16. The engineered extracellular vesicle of any one of claims 1-15, wherein the first fusion protein comprises an amino acid sequence identical to SEQ ID NO: 1 or SEQ ID NO: 13; and the second fusion protein comprises an amino acid sequence identical to SEQ ID NO: 2 or SEQ ID NO: 14.58530.045W012024-123-0217. A composition comprising the engineered extracellular vesicle of any one of claims 1-16, and a pharmaceutically acceptable carrier, excipient, or diluent.

18. A composition comprising a first engineered extracellular vesicle and a second engineered extracellular vesicle, the first engineered extracellular vesicle comprising a first fusion protein comprising the formula A-L1-B-L2-C, wherein A is an anti-CD3 antibody moiety, B is an anti-CLL-1 antibody moiety, and C is a first transmembrane domain;the second engineered extracellular vesicle comprising a second fusion protein comprising the formula D-L3-E-L4-F, wherein D is a PD-1 protein, E is a second transmembrane domain, and F is a CD70 protein;wherein both the first fusion protein and the second fusion protein are displayed on a surface of the first engineered extracellular vesicle and the second engineered extracellular vesicle, respectively; and wherein each of L1- L4comprise an amino acid sequence (GGGGS)n(SEQ ID NO; 50), wherein n is 1, 2, 3, 4, or 5.

19. The composition of claim 18, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

20. A method of treating acute myeloid leukemia (AML) comprising:administering to a subject in need thereof an effective amount of the engineered extracellular vesicle of any one of claim 1-16, wherein the engineered extracellular vesicle treats the AML.59530.045W012024-123-02

Images