WO2020205579A1

WO2020205579A1 - Genetically modified exosomes for immune modulation

Info

Publication number: WO2020205579A1
Application number: PCT/US2020/025374
Authority: WO
Inventors: Yong Zhang
Original assignee: University Of Southern California
Priority date: 2019-03-29
Filing date: 2020-03-27
Publication date: 2020-10-08

Abstract

Extracellular vesicles, such as exosomes, are nano-sized membranous vesicles and are widely distributed in various body fluids. These cell-derived nanoparticles are less immunogenic than artificial delivery vehicles, and engineered forms of extracellular vesicles hold great potential as novel therapeutic modalities. Acute myeloid leukemia (AML) is a common type of leukemia with poor prognosis in adults. In this disclosure, extracellular vesicles, and in particular, synthetic multivalent antibodies retargeted exosomes (SMART-Exos) were genetically engineered to display two distinct types of monoclonal antibodies on the exosomal surface. By targeting CLL-1 and T-cell CD3 receptor, the engineered extracellular vesicles were designed to redirect T cells against AML cells for killings. The anti-CD3×CLL-1 extracellular vesicles. not only bind tightly to both T-cells and CLL-1-positive AML cells, but also elicit potent antitumor immunity in a dose-dependent manner.

Description

GENETICALLY MODIFIED EXOSOMES FOR IMMUNE MODULATION

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62/826,614 filed March. 29, 2019, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on _ , 2020, is named _ SL.txt and is _ bytes in size.

BACKGROUND OF THE INVENTION

For a healthy adult, approximately 10¹¹-10¹² new blood cells are produced daily to maintain a steady state in the peripheral circulation. This remarkable cell renewal process is supported by a small population of bone marrow cells termed hematopoietic stem cells (HSCs). Bone marrow is home to HSCs that may differentiate into any mature blood cell types. The formation of different blood cells from HSCs to mature cells is illustrated in Figure 1.

Leukemia is any myeloid or lymphoid malignancy that develops in the peripheral blood and bone marrow. Patients with leukemia have bone marrow that produces abnormal and poorly functioning white blood cells that divide out of control. The broad classification of leukemia is based on the rapidity of the clinical course, either chronic or acute leukemia. And these two types of leukemia can be further grouped based on the lineage of affected white blood cells. Leukemia that affects lymphoid cells is called lymphoid, lymphocytic, or lymphoblastic leukemia, while the one derived from myeloid cells is classified as myeloid, myelogenous, or myeloblastic leukemia. Thus, depending on whether the leukemia is chronic or acute, myeloid or lymphocytic, there are four main classifications of leukemia:

Chronic lymphocytic leukemia (CLL) occurs when the bone marrow produces a high abundance of lymphocytes. In most cases, CLL progresses slowly and exhibits no symptoms at early stages. This type of leukemia most commonly occurs in adults, and patients diagnosed with this disease most often are over the age of 55. It accounts for nearly 15,000 new cases of leukemia each year.

Chronic myeloid leukemia (CML) is a slow, progressive bone marrow and blood type cancer caused by an increased number of granulocytes. CML is generally diagnosed by detection of a malfunction in two chromosomes resulting in the hybrid creation of another chromosome called the Philadelphia Chromosome. It accounts for around 5,000 new cases of leukemia each year and mainly affects older adults.

Acute lymphocytic leukemia (ALL) is a type of progressive bone marrow and blood cancer caused by the rapid proliferation of immature lymphocytes. The excessive abnormal lymphoid cells eventually crowd out healthy cells in the bone marrow, and metastasize to other organs, which can be fatal in weeks to months if left untreated. The symptoms of AML include anemia, infection, fever, and unexpected bleeding. ALL is most commonly found in childhood and young adulthood. It accounts for around 5,000 new cases of leukemia each year.

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.

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

SUMMARY

This disclosure provides genetically engineered extracellular vesicles, such as synthetic multivalent antibodies retargeted exosomes (SMART-Exos), comprising one or more distinct types of monoclonal antibodies or fragment thereof displayed as a single contiguous polypeptide on the extracellular vesicle surface. Accordingly, the invention provides also an engineered extracellular vesicle comprising a fusion protein that includes consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) an epitope tag, (b) a first antibody moiety, (c) a first peptide linker, (d) a second antibody moiety, (e) a second peptide linker, (f) an extracellular vesicle membrane protein or a portion thereof, and an extracellular vesicle such that the fusion protein is displayed on a surface of the extracellular vesicle.

In some embodiments of the invention, the extracellular vesicle comprises one or more of an exosome, a liposome, a microvesicle, and an apoptotic body. Each of the extracellular vesicle may be isolated from a eukaryotic cell or a prokaryotic cell. In preferred embodiments, the extracellular vesicle is an exosome.

Preferably, the antibody moiety is a single chain variable fragment (scFv), single domain antibody, a bispecific antibody, or multispecific antibody.

The fusion protein may include a first antibody moiety that binds to a T-cell marker protein or a cancer cell surface-marker protein and a second antibody moiety that binds to a T- cell marker protein or a cancer cell surface-marker protein.

In certain embodiments, the T-cell or immune cell marker comprises one or more of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD24, CD25, CD27, CD28, CD30, CD31, CD38, CD40L, CD45, CD56, CD68, CD91, CD114, CD163, CD206, LFA1, PD1, ICOS, BTLA, KIR, CD137, OX40, LAG3, CTLA4, TIM3, B7-H3, B7-H4, , and T-cell Receptor.

In certain embodiments, the cancer cell surface-marker is a cancer cell marker overexpressed by acute myeloid leukemia (AML) cancer cells comprising one or more of CLL- 1, HER2, HER3, EGFR, CD33, CD34, CD38, CD123, TIM3, CD25, CD32, and CD96.

In certain embodiments, the extracellular vesicle membrane protein comprises Platelet Derived Growth Factor Receptor (PDGFR), Lamp2b, lactadherin C1C2 domain, CD13, CD9. In certain embodiments, the membrane protein is a portion of a membrane protein. For example, in certain embodiments, the membrane protein comprises a transmembrane domain of the PDGFR.

In some embodiments, the first antibody moiety comprises a first VL region, a first VH region, and a third peptide linker therebetween. The second antibody moiety comprises a second VL region, a second VH region, and a fourth peptide linker therebetween.

In some embodiments, each of the peptide linkers may include a linker sequence of (GGGS)n (SEQ ID NO: 18) where n is an integer from 1 to 5 or the linker sequence is (GGGGS)n (SEQ ID NO: 19) where n is an integer from 1-5. In preferred embodiments of the invention, the peptide linker is (GGGS GGGS GGGSGGGS peptide (SEQ ID NO: 11) or a GGGGSGGGGSGGGGS peptide (SEQ ID NO: 12).

In some embodiments, the first antibody moiety binds to a T-cell marker protein or a cancer cell surface-marker protein and the second antibody moiety binds to a T-cell marker protein or a cancer cell surface-marker protein.

Preferably the T-cell marker is CD3 and the cancer cell surface- marker is a cancer cell marker overexpressed by acute myeloid leukemia (AML) cancer cells. In one embodiment, the cancer cell surface-marker is CLL-1. In one embodiment, the first antibody moiety binds to a T-cell or immune cell marker protein, the T-cell or immune cell marker protein comprising one or more of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD24, CD25, CD27, CD28, CD30, CD31, CD38, CD40L, CD45, CD56, CD68, CD91, CD114, CD163, CD206, LFA1, PD1, ICOS, BTLA, KIR, CD137, OX40, LAG3, CTLA4, TIM3, B7-H3, B7-H4, , and T-cell Receptor, and the second antibody moiety binds to a cancer cell surface marker protein, the cell surface marker protein comprising one or more of CLL-1, HER2, HER3, EGFR, CD33, CD34, CD38, CD123, TIM3, CD25, CD32, and CD96.

In another preferred embodiment, the first antibody moiety binds to a cancer cell surface marker protein, the cell surface marker protein comprising one or more of CLL-1, HER2, HER3, EGFR, CD33, CD34, CD38, CD123, TIM3, CD25, CD32, and CD96, and the second antibody moiety binds to a T-cell or immune cell marker protein, the T-cell or immune cell marker protein comprising one or more of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD 16, CD24, CD25, CD27, CD28, CD30, CD31, CD38, CD40L, CD45, CD56, CD68, CD91, CD114, CD163, CD206, LFA1, PD1, ICOS, BTLA, KIR, CD137, OX40, LAG3, CTLA4, TIM3, B7-H3, B7-H4, , and T-cell Receptor.

In one embodiment, the first antibody moiety binds to a T-cell marker protein, the T- cell marker protein comprising CD3, and the second antibody moiety binds to a cancer cell surface marker protein, the cell surface marker protein comprising CLL-1.

In another preferred embodiment, the first antibody moiety binds to a cancer cell surface marker protein, the cell surface marker protein comprising CLL-1, and the second antibody moiety binds to a T-cell marker protein, the T-cell marker protein comprising CD3.

In preferred embodiments, the epitope tag comprises hemagglutinin. In some embodiments, the extracellular vesicles have a particle size of about 25 nm to about 150 nm.

In preferred embodiments, each of the first antibody moiety and the second antibody moiety comprises a scFv. Preferably, the first antibody scFv binds to a T-cell marker protein, the T-cell marker protein comprising CD3, and the second antibody scFv binds to a cancer cell surface- marker protein, the cell surface marker protein comprising CLL-1. Alternatively, the first antibody scFv binds to a cancer cell surface marker protein, the cell surface marker protein comprising CLL-1, and the second antibody scFv binds to a T-cell marker protein, the T-cell marker protein comprising CD3.

In certain embodiments, the orientations of the VL and VH domains of the first antibody scFv and the orientations of the VL and VH domains of the second antibody scFv is V_{L- aCD3} - V_{H- aCD3} -V_L-aCLL-1- V_H-aCLL-1 ( aCD3-aCLL-1 scFv)(SEQ ID NO: 40), V_{H- aCD3} - V_{L- aCD3} -V_L- _aCLL-1 -V_H-aCLL-1 (aCD3-aCLL-1 scFv) (SEQ ID NO: 41), V_{H- aCD3} - V_{L- aCD3} -V_H-aCLL-1 -V_L-aCLL- ₁ ( aCD3-aCLL-1 scFv) (SEQ ID NO: 42), or V_{L- aCD3} - -V_{H- aCD3} - -V_H-aCLL-1 -V_L-aCLL-1 ( aCD3-aCLL-1 scFv) (SEQ ID NO: 43).

In certain embodiments, the orientation of the of the VL and VH domains of the first antibody scFv and the orientation of the of the VL and VH domains of the second antibody scFv is V_H-aCLL-1 -V_L-aCLL-1 - V_{L- aCD3} - - V_{H- aCD3} (aCLL-1 - aCD3 scFv) (SEQ ID NO: 44), V_H- _aCLL-1 -V_L-aCLL-1 - V_{H- aCD3} - V_{L- aCD3} (aCLL-1 - aCD3 ScFv) (SEQ ID NO: 45), V_L-aCLL-1 -V_H-aCLL- ₁ - V_{L- aCD3} - V_{H- aCD3} (aCLL-1 - aCD3 scFv) (SEQ ID NO: 46), or V_L-aCLL-1 -V_H-aCLL-1 - V_{H- aCD3} - - V_{L- aCD3} (aCLL-1 - aCD3 scFv) (SEQ ID NO: 47).

One embodiment of the invention comprises engineered extracellular vesicle comprising a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) a hemagglutinin epitope tag, (b) a first antibody scFv that binds to a cancer cell surface marker, the cancer cell surface marker comprising CLL-1, (c) a first peptide linker, (d) a second antibody scFv that binds to a T-cell surface marker, the T-cell surface marker comprising CD3, (e) a second peptide linker, and (f) an extracellular vesicle membrane protein is Platelet Derived Growth Factor Receptor (PDGFR), Lamp2b, lactadherin C1C2 domain, CD13, CD9, and portions thereof, wherein the fusion protein is displayed on a surface of the engineered extracellular vesicle.

Another embodiment comprises an engineered extracellular vesicle comprising a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) a hemagglutinin epitope tag, (b) a first antibody scFv that binds to a cancer cell surface marker, the cancer cell surface marker comprising CLL-1, (c) a first peptide linker, (d) a second antibody scFv that binds to a T-cell surface marker, the T-cell surface marker comprising CD3, (e) a second peptide linker, and (f) an extracellular vesicle membrane protein comprises the transmembrane domain of PDGFR, wherein the fusion protein is displayed on a surface of the engineered extracellular vesicle.

In other preferred embodiment of the invention, the fusion protein includes only one antibody moiety. For example, one certain embodiment of the invention comprises an engineered extracellular vesicle comprising a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) an epitope tag, (b) an antibody moiety, (c) a peptide linker, and (d) an extracellular vesicle membrane protein or portions thereof, wherein the fusion protein is displayed on a surface of the engineered extracellular vesicle. Preferably, the antibody moiety binds to a T-cell or immune cell marker comprising one or more of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD24, CD25, CD27, CD28, CD30, CD31, CD38, CD40L, CD45, CD56, CD68, CD91, CD114, CD163, CD206, LFA1, PD1, ICOS, BTLA, KIR, CD137, OX40, LAG3, CTLA4, TIM3, B7-H3, B7-H4, and a T-cell Receptor. Alternatively, the antibody moiety binds to a cancer cell surface-marker comprising one or more of CLL-1, HER2, HER3, EGFR, CD33, CD34, CD38, CD123, TIM3, CD25, CD32, and CD96.

Preferably, the antibody moiety comprises one or more of a single chain variable fragment (scFv), a single domain antibody, a bispecific antibody, and a multispecific antibody.

Preferably, the extracellular vesicle membrane protein comprises Platelet Derived Growth Factor Receptor (PDGFR), Lamp2b, lactadherin C1C2 domain, CD 13, CD9, or portions thereof.

In one preferred embodiment of the invention, the engineered extracellular vesicle comprising a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) an hemagluttinin epitope tag, (b) single chain variable fragment (scFv) that binds to CLL- 1 , (c) a peptide linker, and (d) an extracellular vesicle membrane protein that comprises a transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR).

In another preferred embodiment of the invention, the engineered extracellular vesicle comprising a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) an hemagluttinin epitope tag, (b) single chain variable fragment (scFv) that binds to CD3, (c) a peptide linker, and (d) an extracellular vesicle membrane protein that comprises a transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR).

Various embodiments also provide for the use of the compositions described herein for use in medical therapy. The medical therapy can be treating cancer, for example, leukemia, breast cancer, lung cancer, pancreatic cancer, prostate cancer, or colon cancer. The invention also provides for the use of a composition as described herein for the manufacture of a medicament to treat a disease in a subject such as a mammal, for example, cancer in a human. The medicament can include a pharmaceutically acceptable diluent, excipient, or carrier.

Various embodiments also provide a method to target cancer cells comprising forming engineered extracellular vesicle comprising a fusion protein that includes consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) an epitope tag, (b) a first antibody moiety, (c) a first peptide linker, (d) a second antibody moiety, (e) a second peptide linker, (f) an extracellular vesicle membrane protein or a portion thereof, and an extracellular vesicle such that the fusion protein is displayed on the surface of the extracellular vesicle. Preferably, the cancer cells are AML cancer cells and the engineered extracellular vesicles selectively target AML cancer cells by binding to one more cancer cell surface markers such as CLL-1 and recruiting T-cells to the cancer cells so that the T-cells selectively kill the cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

Figure 1 illustrates the process of hematopoiesis.

Figure 2 illustrates the categories of extracellular vesicles. Apoptotic bodies (500-2000 nm) are derived from cell undergoing programmed cell death. Microvesicles (100-1000 nm) are produced directly from plasma membrane. Exosomes (30 -100 nm) are generated from late endosomal compartments through fusion of multivesicular bodies with the plasma membrane.

Figure 3 illustrates the biochemical composition of exosomes. Exosomes carry a variety of cellular components including mRNAs, miRNAs and proteins that are often selectively packaged from the host cells. The exosome membrane contains various proteins, including major histocompatibility complexes (MHC I and II), targeting and adhesion (integrins and tetraspanins) molecules, membrane trafficking regulators (annexins and Rab proteins) as well as lipid-rafts.

Figure 4 illustrates the molecular designs of fusion protein inserts for (A) aCD3 SMART-Exos, (B) aCLL-1 SMART-Exos, and (C) aCD3-aCLL-1 SMART-Exos. Single-chain variable fragment (scFv) antibodies were genetically linked to PDGFR transmembrane (TM) domain fusions. A hemagglutinin (HA) epitope tag was fused at the N-terminus of the fusion protein.

Figure 5 illustrates (A) the generated fusion proteins were detected by Western blot assay using an aHA antibody. Lane 1: Native exosomes; Lane 2: aCD3 exosomes ; Lane 3:aCLL-1 exosomes; Lane 4: aCD3-aCLL-1 SMART-Exos. (B) The exosome-specific markers CD9, CD63, and CD81 were detected by Western blot assay. Lane 1: Native exosomes; Lane 2: aCD3 exosomes; Lane 3: aCLL-1 exosomes; Lane 4: aCD3-aCLL-1 SMART-Exos.

Figure 6 illustrates representative negative staining transmission electron microscopy (TEM) images of aCD3-aCLL-1 SMART-Exos.

Figure 7 illustrates NTA measurement of size distribution of aCD3-aCLL-1 SMART-

Exos.

Figure 8 illustrates CLL-1 antigen expression levels on different AML cell lines analyzed by flow cytometry. Black line: U937 cell line; dark gray line (largely behind the black U937 line): HL60 cell line; light gray line (left peak): KG-1A cell line.

Figure 9 illustrates (A) Flow cytometric analysis of the binding of SMART-Exos to Jurkat cells. (B) Flow cytometric analysis of the binding of SMART-Exos to U937 cells. (C) Flow cytometric analysis of the binding of SMART-Exos to HL60 cells. (D) Flow cytometric analysis of the binding of SMART-Exos to KG-1A cells.

Figure 10 illustrates (A) In vitro cytotoxicity of SMART-Exos redirecting healthy PBMCs against U937 after 24 h of incubation at an E:T ratio of 1:10. A mixture of aCD3 and aCLL-1 SMART-Exos was used as a control. Each data point represents a mean of triplicate samples. Error bars are representative of standard deviation. N.A. stands for not applicable. (B) In vitro cytotoxicity of SMART-Exos redirecting healthy PBMCs against HL60 after 24 h of incubation at an E:T ratio of 1:10. A mixture of aCD3 and aCLL-1 SMART-Exos was used as a control. Each data point represents a mean of triplicate samples. Error bars are representative of standard deviation. N.A. stands for not applicable. (C) In vitro cytotoxicity of SMART-Exos redirecting healthy PBMCs against KG-1A after 24 h of incubation at an E:T ratio of 1:10. A mixture of aCD3 and aCLL-1 SMART-Exos was used as a control. Each data point represents a mean of triplicate samples. Error bars are representative of standard deviation. N.A. stands for not applicable.

Figure 11 illustrates in vitro cytotoxicity of aCD3-aCLL-1 SMART-Exos against all three AML cell lines (U937, HL60, and KG-1A) after 24 h of incubation at an E:T ratio of 1:10. Each data point represents a mean of triplicate samples. Error bars are representative of standard deviation. N.A. stands for not applicable.

DETAILED DESCRIPTION

Cell-derived exosomes have shown to be promising nanotechnology platform in recent years. Their ideal size range and intrinsic biocompatibility attract great research interest in developing exosomes as nanomedicine for various diseases. While the majority of studies have focused on the applications of exosomes as therapeutic drug carriers, there is also growing interests in studying whether and to what extant exosomes may be genetically modified for immune modulation. Applicant’s disclose herein a new class of genetically engineered exosomes, termed synthetic multivalent antibodies retargeted exosomes (SMART-Exos). These exosomes are genetically engineered to display two distinct antibodies as a single fusion protein on their surface. The resulting dual targeting SMART-Exos may bind the T-cell CD3 protein, a T-cell surface marker, and the CLL-1 protein, a myeloid lineage-restricted cell surface marker, with the aim of stimulating the immune responses by redirecting and activating T-cells to induce T-cell-mediated tumor cell killing.

Definitions

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 14^th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

Reference herein to "one embodiment", "an embodiment", etc., indicate 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 connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

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.

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.

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.

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 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.

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.

One or ordinary skill 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.

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.

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.

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 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).

The terms "treating", "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and/or prophylactic administration, as appropriate.

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.

As used herein, the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of the compositions of the disclosure into a subject by a method or route which results in at least partial localization of the composition to a desired site. The compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.

The compositions described herein may be administered with additional compositions to prolong stability and activity of the compositions, or in combination with other therapeutic drugs.

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.

The term“substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by aboutl%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

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 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.). 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.

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.

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.

Embodiments of the Disclosure

Embodiments of the engineered extracellular vesicles disclosed herein generally include a fusion protein comprising an (a) an epitope tag, (b) a first antibody moiety, (c) a first peptide linker, (d) a second antibody moiety, (e) a second peptide linker, and (f) an extracellular vesicle membrane protein or portions of the membrane protein such that the fusion protein is displayed on the surface of the engineered extracellular vesicle and selectively bind to a target. In preferred embodiments, the fusion protein, starting from the N-terminus, includes a first antibody moieties that may bind to a specific T-cell marker protein and a second antibody moiety that may bind to a cancer cell surface marker protein. Alternatively, the fusion protein, starting from the N-terminus, includes a first antibody moiety that may bind to a cancer cell surface marker protein and the second antibody moiety may bind to a specific T-cell marker protein.

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 VH and VL regions 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 VH and VL is 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., IgG1, IgG2, IgG3, IgG4, IgA1 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 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.

In certain embodiments, the antibody moieties may comprise one or more of a single chain variable fragment (scFv), single domain antibody, a bispecific antibody, or multispecific antibody. In preferred embodiments, the first and second antibody moieties 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 V_L-linker-VH or may comprise VH- linker- VL. SCFV molecules are known in the art and their production is described, for example, in U.S. Pat. No. 4,946,778, and U.S. Pat. No. 5,641,870.

The term“bispecific antibody” refers to an antibody that shows specificities to two different types of antigens. The term "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 Pat. Pubs. W02014031174 and W02009080252.

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. Pat. Pub. No. 20060002935 Al.

One specific embodiment comprises a fusion protein comprising an (a) an epitope tag, (b) a first antibody single-chain variable fragment (scFv), (c) a first peptide linker, (d) a second antibody scFv, (e) a second peptide linker, (f) and a platelet-derived growth factor receptor transmembrane (PDGFR TM) domain. The fusion protein having the two scFv moieties may be displayed on the surface of an exosome to selectively bind to a target.

One preferred embodiment includes an extracellular vesicle comprises a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) a hemagglutinin epitope tag, (b) a first antibody scFv that binds to a T-cell surface marker, the T-cell surface marker comprising CD3, (c) a first peptide linker, (d) a second antibody scFv that binds to a cancer cell surface marker, the cancer cell surface marker comprising CLL-1, (e) a second peptide linker, and (f) an extracellular vesicle membrane protein comprising the transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR), wherein the fusion protein is displayed on a surface of the engineered extracellular vesicle. The amino acid sequence of this fusion protein is set forth in SEQ ID NO: 13 and the corresponding nucleotide sequence is set forth in SEQ ID NO: 14. In certain embodiments, the amino acid sequence is at least 90%, 95%, or 100% identical to SEQ ID NO: 13.

One preferred embodiment includes an extracellular vesicle comprising a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) a hemagglutinin epitope tag, (b) a first antibody scFv that binds to a cancer cell surface marker, the cancer cell surface marker comprising CLL-1, (c) a first peptide linker, (d) a second antibody scFv that binds to a T-cell surface marker, the T-cell surface marker comprising CD3, (e) a second peptide linker, and (f) an extracellular vesicle membrane protein comprising of the transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR), wherein the fusion protein is displayed on a surface of the engineered extracellular vesicle. The amino acid sequence of this fusion protein is set forth in SEQ ID NO: 15 and the corresponding nucleotide sequence is set forth in SEQ ID NO: 16. In certain embodiments, the amino acid sequence is at least 90%, 95% ,or 100% identical to SEQ ID NO: 15.

When the fusion protein includes, in order from the N-terminus, a first antibody moiety is an scFv moiety that binds to a specific T-cell marker protein (e.g., CD3) and the second antibody moiety is an scFv moiety that binds to a cancer cell surface marker protein (e.g., CLL- 1), the orientation of the VL and VH chains of the first antibody scFv and the orientation of the VL and VH chains of the second antibody scFv is V_{L- aCD3} -V_{H- aCD3} -V_L-aCLL-1-V_H-aCLL-1 ( aCD3- aCLL-1 - scFv)(SEQ ID NO: 40), V_{H- aCD3} - V_{L- aCD3} -V_L-aCLL-1 -V_H-aCLL-1 (aCD3-aCLL-1 scFv)(SEQ ID NO:41), V_{H- aCD3} - V_{L- aCD3} -V_H-aCLL-1 -V_L-aCLL-1 (aCD3-aCLL-1 scFv)(SEQ ID NO:42), or V_{L- aCD3} -V_{H- aCD3} -V_H-aCLL-1 -V_L-aCLL-1 (aCD3-aCLL-1 scFv)(SEQ ID NO: 43). When the fusion protein includes, in order from the N-terminus, a first antibody moiety that is an scFv moiety that binds a cancer cell surface marker protein and the second antibody moiety is an scFv moiety bind to a specific T-cell marker protein, the orientation of the VL and VH domains of the first antibody scFv and the VL and VH domains of the second antibody scFv is V_H-aCLL-1 -V_L-aCLL-1 - V_{L- aCD3} -V_{H- aCD3} (aCLL-1 - aCD3 scFv)(SEQ ID NO:44), V_H-aCLL-1 -V_L-aCLL-1 - V_{H- aCD3} - V_{L- aCD3} ( -aCLL-1 - aCD3 scFv)(SEQ ID NO: 45), V_L-aCLL-1 -V_H-aCLL-1 - V_L- aCD3 -V_{H- aCD3} (aCLL-1 - aCD3 scFv)(SEQ ID NO: 46), or V_L-aCLL-1 -V_H-aCLL-1 - V_{H- aCD3} - V_L- _acD₃ (aCLL-1 - aCD3 scFv)(SEQ ID NO: 47). It is understood that the orientation of the VL and V_H chains include a linker peptide (e.g., (GGGS)₄ (SEQ ID NO: 11) or (GGGGS)₃ (SEQ ID NO: 12) between each of the VL and VH domains of the first antibody moiety and the second antibody moiety. Furthermore, one or more linker peptides may connect the first antibody moiety to the second antibody moiety. For example, for the orientation of the VL and VH domains of (aCD3-aCLL-1 scFv), one or more peptide linkers connects each VL domain to a VH domain of an antibody moiety and also a peptide linker connects a variable domain of the first antibody moiety to a variable domain of the second antibody moiety such that the orientation may be expressed V_{L- aCD3} - linker - V_{H- aCD3} -linker V_H-aCLL-1 - linker -V_L-aCLL-1. Preferably the peptide linker sequence is either (GGGS)n (SEQ ID NO: 18) where n is an integer between 1 and 5 or (GGGGS)n (SEQ ID NO: 19) where n is an integer between 1 and 5. In preferred embodiments of the invention, the peptide linker is ( GGGS )₄ (SEQ ID NO: 11) or (GGGGS)₃ (SEQ ID NO: 12).

In certain preferred embodiments, the ScFv has the amino acid sequence set forth in SEQ ID NO: 20 and binds to the cancer cell surface marker CLL-1. The corresponding nucleotide sequence is set forth in SEQ ID NO: 21. In various embodiments, the amino acid sequence of the scFv that binds to CLL-1 is at least 90%, 95%, or 100% identical to SEQ ID NO: 20.

In other preferred embodiments, the ScFv has the amino acid sequence set forth in SEQ ID NO: 22 and binds to the T-cell marker CD3. The corresponding nucleotide sequence is set forth in SEQ ID NO: 23. In certain embodiments, the amino acid sequence of the scFv that binds to CD3 is at least 90%, 95%, or 100% identical to SEQ ID NO: 22.

In other embodiments, the scFv that binds to CD3 includes the VL domain and VH domain set forth in SEQ ID NO: 24 and SEQ ID NO:26, respectively. The corresponding nucleotide sequences of the VL domain and VH domain are set forth in SEQ ID NO: 25 and SEQ ID NO:27, respectively. In some embodiments, the amino acid sequence of the VL domain is at least 90%, 95%, or 100% identical to SEQ ID NO: 24 and the amino acid sequence of the VH domain is at least 90%, 95%, or 100% identical to SEQ ID NO: 26.

In other embodiments, the scFv that binds to CLL-1 includes the VL domain and VH domain set forth in SEQ ID NO: 30 and SEQ ID NO:32, respectively. The corresponding nucleotide sequences of the VL domain and VH domain are set forth in SEQ ID NO: 31 and SEQ ID NO:33, respectively. In some embodiments, the amino acid sequence of the VL domain is at least 90%, 95%, or 100% identical to SEQ ID NO: 30 and the amino acid sequence of the VH domain is at least 90%, 95%, or 100% identical to SEQ ID NO: 32.

In certain embodiments, the fusion protein further comprises an exosomal surface protein or a portion thereof, to anchor the fusion protein on the surface of the exosome. Exemplary exosomal surface proteins that my form part of the fusion protein includes platelet- derived growth factor receptor beta (PDGFR), Lam2b, lactadherin C1C2 domain, CD13, CD82, CD81, CD63, CD86, and CD9. In preferred embodiments, the exosomal surface protein incorporated into the fusion protein is platelet derived growth factor receptor beta (PDGFR) (RefSeq: NM_002609, NP_002600). Platelet-derived growth factor receptors are cell surface tyrosine kinase receptors for members of the platelet-derived growth factor (PDGF) family. These exosomal membrane protein or portions thereof maybe used to express protein on cell membrane surface, and in particular, on the surface of an exosome. Preferably, the fusion protein incorporates the single transmembrane domain of PDGFR and is at least 50%, at least 60%, at least 75%, at least 90%, at least 95%, or 100% identical to the amino acid sequence

Methods of creating fusion proteins are disclosed, for example, in U.S. Pat. No. 6,403,769 and are well known in the art.

In certain preferred embodiments, the T-cell or immune cell marker protein may comprise, for example, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD24, CD25, CD27, CD28, CD30, CD31, CD38, CD40L, CD45, CD56, CD68, CD91, CD114, CD163, CD206, LFA1, PD1, ICOS, BTLA, KIR, CD137, OX40, LAG3, CTLA4, TIM3, B7-H3, B7- H4, , and the T-cell Receptor. Preferably, the T-cell marker protein is CD3. The CD3 (cluster of differentiation 3) molecule is encoded by the gene mapped to human chromosome 1 lq23.3. The CD3 protein exists in three isoforms, CD3e, CD3g and CD3d and each contains an N- terminal extracellular domain, a transmembrane segment and a cytoplasmic domain. CD3 is a 20kDa glycoprotein expressed on the surface of all human T lymphocytes. Human CD3 e nucleotide sequence is illustrated by the GenBank accession number NM_000733. Human CD3g nucleotide sequence is illustrated by the GenBank accession number NM_000073. Human CD3 d nucleotide sequence is illustrated by the GenBank accession number NM_000732. The amino acid sequence for human CD3e, CD3d, and CD3g, is illustrated by Genbank accession numbers NP_000724, NP_000723, and NP_000064, respectively, and the murine amino acid sequences of CD3e, CD3d, and CD3g is illustrated by Genbank accession numbers NP_031674, NP_038515, and NP_033980, respectively.

In certain preferred embodiments, the cancer cell surface marker may comprise a cancer cell surface marker overexpressed by acute myeloid leukemia (AML) cancer cells. These cancer cell surface markers may include, for example, CLL-1, HER2, HER3, EGFR, CD34, CD38, CD123, TIM3, CD25, CD32, and CD96.

In certain preferred embodiments, the cancer cell surface marker is CLL-1. CLL-1 refers to C-type lectin-like molecule- 1, which is an antigenic determinant detectable on leukemia precursor cells and on normal immune cells. C-type lectin-like-1 (CLL-1) is also known as MICL, CLEC12A, CLEC-1, Dendritic Cell-Associated Lectin 1, and DCAL-2. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CLL-1 can be found as UniProt/Swiss-Prot Accession No. Q5QGZ9 and the nucleotide sequence encoding of the human CLL-1 can be found at Accession Nos. NM 001207010.1, NM 138337.5, NM 201623.3, and NM 201625.1.

Peptide linker groups may be used to connect various portions of the fusion protein, for example, between an scFv cancer cell surface binding moiety and the PDGFR transmembrane domain or between variable heavy and variable light chain of the scFv molecule. Preferably, the linker sequence is either (GGGS)n (SEQ ID NO: 18) where n is an integer between 1 and 5 or (GGGGS)n (SEQ ID NO: 19) where n is an integer between 1 and 5. Preferably, the(GGGS)n (SEQ ID NO: 18) linker sequence is a (GGGS)4 peptide (SEQ ID NO: 11) and the (GGGGS)n (SEQ ID NO: 19) linker sequence is a (GGGGS)₃ peptide (SEQ ID NO: 12). In certain embodiments, the first, second, third and fourth peptide linkers are one or more of a(GGGS)₄ peptide (SEQ ID NO: 11) or (GGGGS)₃ peptide (SEQ ID NO: 12). The linker sequence may be varied depending on the polypeptide portions to be linked to form the fusion protein. Additional peptide linkers and tags are known in the art, such as 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, 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). Further examples include poly(L-Gly), (Poly L-Glycine linkers); poly(L-Glu), (PolyL-Glutamine linkers); poly (1-Lys), (Poly L- Lysine linkers). In one embodiment, the peptide linker has the formula (amino acid) n, where n is an integer between 2 and 100, preferably wherein the peptide comprises a polymer of one or more amino acids. 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 (SEQ ID NO. 17) disposed at the N- terminus of the fusion protein.

Certain embodiments provide methods of treating a cancer comprising administering to a patient an effective amount of any one of the embodiments of engineered extracellular vesicle described herein. Examples of cancers that may be treated by the current invention includes, but is not limited to, leukemias including acute leukemias and chronic leukemias such as acute lymphocytic leukemia (ALL), Acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML) and Hairy Cell Leukemia; lymphomas such as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotrophic virus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), Hodgkin's disease and non-Hodgkin's lymphomas.

In more specific embodiments, a method for targeting a cancer cell comprises providing a fusion protein that includes consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) an epitope tag, (b) a first antibody moiety, (c) a first peptide linker, (d) a second antibody moiety, (e) a second peptide linker, (f) an extracellular vesicle membrane protein or a portion thereof, and contacting a subject comprising cancer cells and healthy cells with the engineered extracellular vesicle such that the extracellular vesicle selectively targets the cancer cells.

Alternatively, a subject having healthy cells and cancer cells may be administered a therapeutically effective amount of a fusion protein that includes consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to corresponding to (a) an epitope tag, (b) a first antibody single-chain variable fragment (scFv), (c) a first peptide linker, (d) a second antibody scFv, (e) a second peptide linker, (f) a platelet-derived growth factor receptor transmembrane (PDGFR TM) domain, and an extracellular vesicle such that the fusion protein is displayed on a surface of the extracellular vesicle, and the extracellular vesicle selectively targets the cancer cells. 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 mAh 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. Pat. No.6287569 to Kipps el al.) . The amplified cDNAs encoding mAh 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 maybe 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 preferred embodiments, the scFv molecules are genetically fused to the polynucleotide sequence of a hemagglutinin epitope tag and a transmembrane segment of PDGFR.

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.

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).

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.

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 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.

In one embodiment, the present invention provides a method of producing a protein. The method includes transforming a host cell with an expression construct and culturing the host cell under conditions suitable for producing the conjugate in various embodiments, the expression construct includes a nucleic acid molecule encoding a protein conjugate including an scFv and a peptide, wherein the fusion protein includes an amino acid sequence at least 90%, at least 95% or 100% identical to one or more SEQ ID NO: 13 and SEQ ID NO: 15.

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 pRIT2T™. 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 lacI, lacZ, T3, I7, apt, lambda PR, PL, trp, CMV immediate early, HSV thymidine kinase, early and late SV40, retroviral LTR, and mouse metallothionein-I regulatory regions.

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. Discussion

Chemotherapy is the primary treatment for people with AML and characterized by a cure rate between 20-75% for patients younger than 60 years old. This wide range depends primarily on leukemia-cell cytogenetics, which is related to the structure and function of the cell, especially the chromosomes. Usually, once a patient has been in remission for three years, the likelihood of relapse declines sharply to less than 10%. At best, standard approaches of AML treatment can achieve ultimate curative value in 40% of patients. For certain subtypes, however, the curative potential is far from satisfactory level. In patients older than 60 years, chemotherapy results in a cure rate of less than 10%, due to the inability of elderly patients to survive the treatment and they are likely to exhibit therapeutic resistance or have medical impediments to the successful completion of such regimens.

Although chemotherapy can improve overall survival in patients with AML, the prognosis is still poor with a five-year survival rate of 30%, regardless of receiving hematopoietic stem cell transplantation (HSCT). Thus, there is an urgent need to develop new therapeutic approaches for AML treatment.

Hematopoietic stem cell transplantation (HSCT) has been used for the treatment of AML for decades. It involves the infusion of hematopoietic stem cells to reestablish bone marrow function in cancer patients whose bone marrow is removed by receiving bone-marrow- toxic doses of cytotoxic drugs. In AML disease, there is a high incidence of relapse, which has prompted the application of post-remission strategies using either patients' own stem cells (autologous HSCT) or stem cells from another acceptable donor (allogeneic HSCT). Although HSCT has cured patients with hematologic malignancies and bone-marrow damages, it is much more toxic than chemotherapy and immunosuppressive therapy. Moreover, relapse after allogeneic HSCT does occur, and the vast majority of elderly patients are not eligible for HSCT. Therefore, HSCT is only suggested for cases in which the survival time and quality of life exceed that of treatments other than HSCT and should be carefully evaluated in terms of the latest guidelines and transplantation outcomes for each patient.

Traditional treatments for AML involve chemotherapy and radiation, which often cause long-term side effects and multidrug resistance. The nanotechnology has become a powerful approach to overcome limitations associated with conventional drugs. Nanoparticles can enhance the therapeutic efficacy of anticancer agents, improve biocompatibility and delivery, and help overcome treatment resistance. The nanosize range of these particles allows them to cross biological barriers more effectively that may be further improved by functionalizing the nanoconstructs’ surface with specific ligands for precise delivery to the disease targets . In general, the nanosized particles allow for efficient uptake by a variety of cell types and selectively deliver anticancer agents to target sites. There is a wide variety of nanoparticles, including organic, inorganic, and hybrid nanoparticles. Organic nanocarriers have been extensively explored in cancer, including dendrimers, lipid-based nanoparticles, and polymeric nanoparticles. Dendrimers are highly branched, exhibiting high versatility and functionality in drug delivery with a maximum of 10 nm of size. Lipid-based nanoparticles, such as liposomes, micelles, and hybrid systems are prominent drug delivery vehicles with improved biocompatibility and prolonged blood circulation, and typically have 50-100 nm of size. Polymeric nanoparticles, ranging from 10 to 400 nm, are produced from natural, synthetic, hydrolytically, or enzymatically degradable polymers onto which a cytotoxic drug can be covalently attached, dissolved, encapsulated, or entrapped.

Immunotherapy has radically revolutionized cancer therapy over the past decade. Though HSCT is one of the most successful immunotherapeutic strategies for postremission therapy in AML, relapse after allogeneic HSCT does occur, and it is not eligible for most elderly patients. Alternative AML immunotherapies have been studied in the past few years. However, the slow progression of translating immunotherapeutics for AML to the clinic is hindered by the complexity of the disease, including heterogeneous antigen expression of diverse AML cell populations, low endogenous immune responses, and intrinsic immune response-driven resistance mechanisms of the leukemic blasts. Therefore, new immunotherapeutic strategies for AML are urgently needed to improve patients’ survival of this aggressive disease.

At the current stage, various therapeutic modalities have been developed for AML immunotherapy, including targeted immunotherapy, checkpoint inhibitors, therapeutics vaccines, antibody-drug conjugates (ADCs), and chimeric antigen receptor-T cells (CAR-T) therapies. For targeted immunotherapy, it relies on a suitable target antigen to minimize unwanted on-target off-tumor toxicity. In AML, it is difficult to find a lineage-restricted target antigen with a minimal expression on healthy hematopoiesis cells. It is expected that targeting AML-associated antigens will result in boosting the ability of immune cells to kill cancer cells.

Checkpoint inhibitors rely on the improvement of endogenous immune responses by blocking signaling pathways that stop the immune system from attacking the cancer cells. They have been successfully approved in several solid organ malignancies and are now entering the treatment of hematological diseases.

Priming the immune system with therapeutic vaccines, particularly studies based on dendritic cells, have also been shown to induce anti-leukemic immune responses reliably ^[19]. Immune checkpoint blockade therapy and dendritic cells vaccines appear to be safe but have yet to demonstrate their clinical potency when used as a monotherapy for the treatment of AML.

Antibody-drug conjugates (ADCs), consist of monoclonal antibodies conjugated to small-molecule cytotoxic drugs, show great therapeutic potential in AML. Several clinical trials have been performed to evaluate the risk-benefit ratio. In contrast, T cell recruiting antibodies and CAR-T cell constructs are still in the early clinical development for the therapy of AML. Their feasibility of applications and potential side effects have been studied under currently ongoing phase I trials. Future efforts have to be taken to integrate best immunotherapeutic approaches into individualized curative treatment for AML patient.

Biochemical Composition of Extracellular Vesicles

There are various types of extracellular vesicles (EVs). EVs can be mainly categorized in to three classes based on their size and biogenesis pathways: apoptotic bodies, microvesicles, and exosomes. Apoptotic bodies are generally larger in size (500-2000 nm) and are derived from cell undergoing programmed cell death. Microvesicles are membranous vesicles (100- 1000 nm) that bud directly from plasma membrane. Exosomes are lipid bilayer-enclosed nano sized EVs, ranging from 30 to 100 nm in diameter, are secreted throughout all stages of the cell cycle (Figure 2). They are produced from inward budding of endosomal compartments called multivesicular bodies (MVBs) and are released into the extracellular space upon fusion of the MVBs with the plasma membrane. As endogenous nanocarriers, exosomes play important roles in mediating intercellular communication.

Owing to their endosomal origin, several members of the tetraspanin family including CD9, CD63, and CD81 are enriched on exosomal membranes and serve as unique marker proteins (Figure 3). Exosomes contain proteins required for membrane transport and fusion (Rab proteins, annexins, flotillin), proteins associated with MVB biogenesis (Alix, TSG101), and heat shock proteins (Hsc70, Hsp90) as previously reported. This type of vesicles also carries a variety of cellular proteins, RNA and miRNAs, cytoskeletal proteins and metabolic enzymes. In addition, the exosomal membrane is enriched with lipid-rafts including cholesterol, sphingolipids, and ceramide. Interestingly, exosomes secreted from antigen- presenting cells such as dendritic cells express functional major histocompatibility complexes (MHC I and II) on their surface. Exosomes as Next-Generation Cancer Therapy

Extracellular vesicles, and in particular, exosomes hold great therapeutic potential for cancer therapy. The tetraspanins on the exosomal surface can promote direct membrane fusion and facilitate the release of their soluble cargoes into the cytosol. Furthermore, CD47 found on exosomes is shown to prevent exosomes from phagocytosis by circulating monocytes and macrophages and prolong exosomes half-life in the blood circulation. Current nanoparticle delivery systems are confronting with many issues such as off-target cytotoxicity, poor biocompatibility, and low efficacy. Compared with traditionally synthesized nanoparticles and viral vectors, cell-derived exosomes may exhibit significantly reduced immunogenicity while possessing intrinsic targeting properties. Exosomes may cross biological barriers and deliver their cargoes to recipient cells with high selectivity.

Various anticancer drugs and functional proteins have been packed into exosomes for cancer treatment. There are several ways to load cargoes into exosomes. After incubation small membrane-permeable agents together with exosomes, the agents can be passively loaded into exosomes. However, to load membrane-impermeable drugs and functional proteins, such as miRNAs, siRNAs, and small DNAs, electroporation is required to create pores on exosome lipid bilayer membrane to allow them to be incorporated within exosomes. Another approach is surface engineering of exosomes via membrane proteins. The transmembrane protein on the surface of exosomes can be used as a fusion partner.

To deliver protein cargoes, researchers have fused soluble cargoes, such as transcription factors or cytosolic proteins, with membrane proteins of exosomes, aiming to alter or supplement the biological pathways of recipient cells. And engineering tissue-specific ligands on the exosomal surface can enable targeted delivery of drugs and RNA therapeutics to specific target cells.

AML is a common type of leukemia affecting adults. Although standard chemotherapies can improve overall survival in patients with AML, a majority of patients eventually relapse, with a five-year survival rate of 30%. Thus, there is an urgent need to develop new therapeutic approaches for AML treatment.

CLL- 1 is a type II transmembrane glycoprotein, and it is a myeloid lineage-restricted cell surface marker. Importantly, CLL-1 is overexpressed in both AML blasts and leukemia stem cells, but extremely low expression in healthy hematopoietic stem cells (HSCs), which presents a promising therapeutic target for AML treatment.

Exosomes have emerged as attractive nanomedicine platforms in recent years. They offer excellent advantages as delivery systems, owing to their nano-sized particles, low immunogenicity, and long-term safety. Moreover, they are highly versatile in terms of their surface engineering and cargo encapsulation.

Herein we explored using exosomes for immune-modulatory therapy with the aim of genetically engineering exosomes to elicit antitumor immunity against AML cells. Exosomes were genetically engineered to display two individual functional monoclonal antibodies on the exosomal surface for selectively recruiting cytotoxic T cells to cancer cells. The resulting aCLLl-aCD3 SMART-Exos showed significant binding CLL-1⁺ cell lines and potent and selective in vitro cytotoxicity against various AML cell lines.

The antitumor efficacies of SMART-Exos may be determined by a multitude of parameters, including E:T ratio, antigen expression level, cytogenetics, and heterogeneity of patients.

As is shown herein, the dual targeting SMART-Exos exhibit excellent selectivity in inducing potent anticancer immunity against CLL-1 -positive cells, highlighting SMART-Exos as promising candidates for AML immunotherapy.

As described herein, the exosomal surface can be genetically engineered to display various functional membrane proteins. This engineering ability of exosomes provides a versatile platform for the development of exosomes-based therapeutics. But the potency of functional protein displayed on the exosomal surface could be affected by various factors, including the identification of a surface protein to serve as anchoring scaffold, and the configuration of functional peptides. For example, in our case, different orientation of individual antibodies in fusion protein may have different potential elfects on physicochemical and biological properties of SMART-Exos. Other embodiments may include other orientations of variable regions as follows: V_H-aCLL-1 -V_L-aCLL-1 - V_{L- aCD3} - V_{H- aCD3} (aCLL-1 - aCD3 scFv)(SEQ ID NO: 44); V_H-aCLL-1 -V_L-aCLL-1 - V_{H- aCD3} -V_{L- aCD3} (aCLL-1 - aCD3 scFv)( SEQ ID NO: 45) ; V_L-aCLL-1 -V_H-aCLL-1 - V_{L- aCD3} -V_{H- aCD3} (aCLL-1 - aCD3 scFvX SEQ ID NO: 46); V_L- _aCLL-1 -V_L-aCLL-1 - V_{H- aCD3} -V_{L- aCD3} ( -aCLL-1 - aCD3 SCFv)( SEQ ID NO: 47), V_{L- aCD3} -V_{H- aCD3} -V_L-aCLL-1-V_H-aCLL-1 (aCD3-aCLL- 1- scFv)( SEQ ID NO: 40), V_{H- aCD3} -V_{L- aCD3} V_L-aCLL-1 -V_H- _aCLL-1 (aCD3-aCLL-1 scFv)( SEQ ID NO: 41); V_{H- aCD3} -V_{L- aCD3} -V_H-aCLL-1 -V_L-aCLL-1 (aCD3- aCLL-1 scFv)( SEQ ID NO 42); V_{L- aCD3} -V_{H- aCD3} -V_H-aCLL-1 -V_L-aCLL-1 (aCD3-aCLL-1 scFv) (SEQ ID NO: 43).

Exosomes can also be used as carriers for the therapeutic delivery of various synthetic and biological molecules. The efficiencies of current approaches for drug delivery, such as synthesized nanoparticles, vims-like vectors, and proteoliposomes, can still be limited by endosomal entrapment. In contrast, fusogenic exosomes loaded with therapeutic cargoes can deliver therapeutic agents by directly enter the cytosol of targeted cells via fusion, which bypasses the potential for becoming entrapped in an endosome. It is contemplated that the SMART-Exos may be loaded with therapeutic cargos for targeted delivery with enhanced efficacy. Therapeutic cargos may include an allergen, adjuvant, antigen, immunogen, antibody, hormone, cofactor, metabolic enzyme, immunoregulatory enzyme, interferon, interleukin, gastrointestinal enzyme, an enzyme or factor implicated in hemostasis, growth regulatory enzyme, vaccine, antithrombolytic, toxin, antitoxin, a single- stranded or double-stranded oligonucleotide such as .a single-stranded or double-stranded DNA, iRNA, siRNA, mRNA, ncRNA, antisense RNA, miRNA, LNA, morpholino oligonucleotide, or analog or conjugate thereof, or a diagnostic or imaging agent.

Pharmaceutical Formulations

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 b- glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

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.

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.

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.

The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com 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.

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.

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. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and/or gelatin.

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.

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.

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.

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.

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 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith el al.). 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.

Useful dosages of the compositions 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.

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 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.

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 or composition described herein formulated in such a unit dosage form.

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/m² of 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·

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.

The compounds described herein can be effective anti-tumor agents and have higher potency and/or reduced toxicity as compared to known treatments for AML. Preferably, compounds or compositions described herein are more potent and less toxic than known treatments, and/or avoid a potential site of catabolic metabolism encountered with known treatments, i.e., have a different metabolic profile than known treatments.

The invention provides therapeutic methods of treating cancer in 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 various type of malignant neoplasm, for example, colon cancer, breast cancer, melanoma and leukemia, and in general is characterized by an undesirable cellular proliferation, e.g., unregulated growth, lack of differentiation, local tissue invasion, and metastasis.

The ability of a compound or composition described herein to treat cancer may be determined by using assays well known to the art. For example, the design of treatment protocols, toxicity evaluation, data analysis, quantification of tumor cell-kill, and the biological significance of the use of transplantable tumor screens are known. In addition, ability of a compound to treat cancer may be determined using the Tests as described below.

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.

EXAMPLES

Example 1. Materials and Methods

Materials. Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, were purchased from Corning Inc. (Coming, NY). Fetal bovine serum (FBS) was purchased from VWR International (Radnor, PA). Opti-modified Eagle's medium (Opti-MEM), Expi293 expression medium and ExpiFectamine 293 transfection reagent were purchased from Thermo Fisher Scientific (Waltham, MA). Carboxyfluorescein succinimidyl ester (CFSE) and FITC anti-human CD371 (CLEC12A) antibody were purchased from BioLegend (San Diego, CA). Pierce Coomassie Plus (Bradford) assay kit was purchased from Thermo Fisher Scientific (Waltham, MA).

Cell lines. Expi293F cells are suspension cells derived from human HEK293 cell line and were purchased from Thermo Fisher Scientific (Waltham, MA). The Expi293 expression medium is a chemically defined serum- free medium, developed for the growth and transfection of Expi293F cells. This cell line is maintained in the cell medium with shaking at a speed of 125 rpm/min at 37°C in 8% CO2. U937, HL-60, KG-1A, and Jurkat cell lines were all obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C in 5% CO2. Human peripheral blood mononuclear cells (PBMCs) were purchased from HemaCare (Van Nuys, CA).

Molecular cloning of SMART-Exos. The genes encoding scFv fragments of aCLL-1

(1075.7) and aCD3 (UCHT-1) antibodies were inserted into the pDisplay vector (Thermo Fisher Scientific, Waltham, MA). (GGGS)₄ (SEQ OD NO: 11) and (GGGGS)₃ (SEQ ID NO: 12) linkers were inserted between VL (SEQ ID NO: 24) and VH regions (SEQ ID NO: 26) of aCD3 scFv and aCLL-1 scFv, respectively. To make aCD3-aCLL-1 scFv fusion protein, a (GGGGS)3 (SEQ ID NO: 12) flexible linker was designed between two distinct scFv fragments by overlap extension polymerase chain reactions (PCR). The aCD3-aCLL-1 scFv fusion protein is genetically linked to platelet-derived growth factor receptor (PDGFR) transmembrane (TM) domain on the surface of exosomes to form the fusion protein having the amino acid sequence set forth in SEQ ID NO: 13. The orientation of variable region for designed construct is arranged as V_{L- aCD3} -V_{H- aCD3} -V_L-aCLL-1-V_H-aCLL-1 . In addition, aCD3 scFv antibodies(SEQ ID NO: 22) and aCLL-1 scFv antibodies (SEQ ID NO: 20) were separately fused with PDGFR TM domain (SEQ ID NO: 9) for generation of monoclonal exosomes as controls. An N-terminal HA tag (SEQ ID NO: 17) was added for all the antibody - PDGFR TM domain fusions.

Primers used for PCRs to amplify these gene fragments are listed in Table 1. with highlighted restriction enzyme sites of Bglll and Sail.

Table 1. List of primer sequences used for molecular cloning (Bglll restriction site underlined; Sail restriction site in bold).

PCR. To generate gene fragments for cloning, AccuPrime™ Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA) was used for the PCR amplification. The condition for a 50 ml PCR reaction were as follows: Template: 1 ml (50 ng); Forward primer (10 mM): 1.5 ml ;Reverse primer (10 mM): 1.5 ml; 10X AccuPrime™ PCR Buffer: 5 ml; AccuPrime™ Taq DNA Polymerase: 0.5 ml; ddH20: 38 ml.

PCR and overlap extension PCR were followed by steps according to Table 2 and Table 3.

Table 2. PCR Cycle

Table 3. Overlap Extension PCR

Restriction enzyme digestion. The amplified inserts and pDisplay vector were digested by restriction enzymes Bglll and Sail (New England Biolabs, Ipswich, MA). The digestion of DNA fragments was carried out under the conditions recommended by manufacturers. The digested products were ligated between the N-terminal signal peptide and the transmembrane domain of human platelet-derived growth factor receptor (PDGFR) in pDisplay vector by using T4 DNA ligase (New England Biolabs, Ipswich, MA). A mixture of the digested pDisplay vector and insert gene fragments were incubated at 16°C overnight. Plasmid transformation. The generated expression plasmids were transformed into E. coli (strain DH10B). After electroporation, DNA mixed with competent cells were recovered in LB for 1 h and was spread onto a pre- warmed LB agar plate, containing appropriate selective antibiotic (100 mg/ml penicillin), and was incubated at 37°C for the colonies to grow.

Colony PCR. OneTaq DNA Polymerase (New England Biolabs, Ipswich, MA) was used in colony PCR to screen the target colony. Single colonies were picked and resuspended in 10 pi of Taq polymerase mixture to perform PCR. The positive recombinant plasmids screened by colony PCR were confirmed by DNA sequencing provided by GENEWIZ (South Plainfield, NJ).

Agarose gel electrophoresis. Agarose gel electrophoresis was used for analysis of PCR products. 1.5 % agarose gel was used to separate DNA fragments. The agarose gel was prepared by mixing agarose powder with lx TAE buffer to the desired concentration and then heated until completely melted.

DNA gel recovery. Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Irvine, CA) was used to recover DNA fragments from agarose gel. The excision and recovery of the DNA fragments from agarose gel were carried out by the protocols provided by the manufacturer.

Plasmid purification. Sequence-verified expression plasmids were isolated from bacteria culture either by small-scale purification or large-scale purification. The ZR Plasmid Miniprep-Classic kit (Zymo Research, Irvine, CA) was used for small scale plasmid purification. Plasmid-bearing bacteria were inoculated in LB medium containing antibiotic one day prior to plasmid purification. After 12-16 h of incubation at 37°C and shaken at 250 rpm, the bacteria were centrifugated at 4000 xg for lOmin at 4°C to form compacted pellets. The plasmids were then extracted and purified as described in the instruction provided by the manufacturer.

ZymoPURE™ II Plasmid Maxiprep Kit (Zymo Research, Irvine, CA) was used for large scale plasmid purification. Plasmid-bearing bacteria were inoculated in LB medium containing antibiotic and incubated at 37°C for 12-16 h to make a starter culture. 5 ml of starter culture was added to 150 ml of LB medium and was incubated at 37°C, shaken at 250 rpm for another 12 h. The bacteria were harvested by centrifugation at 4000 xg for 10 min at 4°C. The plasmids were then purified by following the manufacturer's instruction.

Expression of SMART-Exos. The expression constructs of SMART-Exos were transfected into Expi293F cells cultured in chemically defined Expi293 expression medium by using ExpiFectamine 293 transfection kits under the manufacturer's protocol. Media were harvested on day 3 and day 6 post-transfection through centrifugation.

Exosomes purification. Engineered exosomes were purified from the harvested culture media through differential centrifugation. Cell culture media were centrifuged at a low speed of 500 xg at 4°C for 10 min to remove detached cells and then 30 min at 4000 xg, followed by 15,000 xg for 50 min to remove cell debris and large vesicles. The obtained supernatants were then centrifugated at 371,000 xg in a Type 70 Ti rotor (Beckman Instruments, Indianapolis, IN) for 2 h at 4°C. After removing supernatant, exosomes were washed and resuspended in PBS, followed by filtration through a 0.22 pm syringe filter. The protein concentrations of purified exosomes were determined by Bradford assays.

Nanoparticle tracking analysis (NT A). Particle concentration and size distribution of the purified exosomes were determined by NTA using a Nanosight LM10 (Malvern Instruments, U.K.) according to the manufacturer's instruction.

Transmission electron microscopy (TEM). The exosomes were prepared and imaged by a JEOL 2010F TEM (JEOL, Peabody, MA). The TEM grids were preincubated with 20 mL of the 0.1% poly-lysine solution for 10 min. Excess liquid was removed with filter paper. 20 mL of the exosomes sample was placed on 200 pm mesh grids and incubated for 15 min. Residual liquid was removed and dried again from the grids with filter paper, followed by staining with 20 mL of 2% uranyl acetate solution for 5 min. The grid was left to air dry.

Western blot analysis. Exosome aliquots containing 3 pg protein were reduced with 10 mM dithiothreitol and boiled at 98 °C for 10 min. For western blot without protein reduction (tetraspanins CD81 and CD63), DTT was omitted and its volume was replaced by PBS. The samples were then separated in 4-20% ExpressPlus-PAGE gels (GeneScript, Piscataway, NJ) at 155 V for 45 min. The gel was carefully removed, washed with buffer, and subsequently transferred to Immun-Blot PVDF membranes (BioRad Laboratories, Inc, Hercules, CA) at 16 V for 30 min using a Trans-Blot SD SemiDry Transfer Cell (Bio-Rad Laboratories, Inc, Hercules, CA). The resulting membranes were blocked with 5% BSA in PBST for 1 h at room temperature while gently shaking. The membranes were incubated with the following primary antibodies: mouse monoclonal anti-HA (clone: 2-2.2.14) from Thermo Fisher Scientific, mouse monoclonal anti-CD63 (clone: H5C6) from BioLegend, mouse monoclonal anti-CD81 (clone: 1.3.3.22) from Thermo Fisher Scientific, and rabbit monoclonal anti-CD9 (clone: D3H4P) from Cell Signaling Technology) for 1 h at room temperature. The membranes were washed (3 x PBST, 5 min) and incubated with secondary antibodies anti-mouse IgG-HRP (catalog number: 62-6520) from Thermo Fisher Scientific and anti-rabbit IgG-HRP (catalog number: 65-6120) obtained from Thermo Fisher Scientific, and further diluted in blocking buffer (1:2000) for 1 h at room temperature. SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific) was used to develop blots according to manufacturer's instructions and immuno-active bands were detected by a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc, Hercules, CA).

Flow cytometric analysis. Cell-based binding assays, antigen expression analyses, and in vitro cytotoxicity assays were performed using flow cytometry. The binding of SMART- Exos to AML cell lines (U937, HL60 and KG-1A) and CD3-positive Jurkat cells were analyzed by flow cytometry. Cells were incubated with 0.1 mg/mL exosomes in ice-cold PBS containing 1% (w/v) BSA and 10% human serum for 30 min on ice and washed with the same medium, followed by incubation with the anti-HA antibody (clone: 2-2.2.14) from Thermo Fisher Scientific for 30 min on ice. After that, cells were washed again and subsequently incubated with the Alexa Fluor 488-labeled goat anti-mouse IgG H&L antibody (catalog number: ab 150113) from Abeam for 30 min on ice. Then the cells were washed, and the binding was evaluated by a BD LSR II Flow Cytometer (BD Biosciences, San Jose, CA). Data were processed by FlowJo_V10 software (Tree Star Inc., Ashland, OR). Cell-bound fluorescent- labeled antibody was analyzed as the mean fluorescence intensity (MFI) for 10 000 cellular events, and whole cells were analyzed using appropriate scatter gates to exclude cellular debris and aggregates. Background fluorescence were determined by using target cells incubated under the same conditions.

For in vitro cytotoxicity assays, target cells stained with CFSE (30,000/well) were mixed with PBMCs (150,000/well) to afford an E:T ratio of 5:1, and incubated with PBS, different concentrations of SMART-Exos and mixtures of monoclonal exosomes for 24 h at 37°C and 5% CO2. Cells were then centrifuged, resuspended in PBS (with 2% FBS), and analyzed with the BD LSR II flow cytometer. Cells that were FITC⁺ (CFSE) were considered as the viable target cells, and the relative viabilities of all treatment groups were normalized to the PBS group.

Example 2. SMART-Exos Characterization and Assays

Plasmid construction. SMART-Exos were generated by displaying two individual antibodies on the exosomal surface with the aim of redirecting the cytotoxic activity of effector T cells to attack cancer cells by targeting T cell CD3 and CLL-1 simultaneously with high specificity. CD3 is an essential T cell co-receptor and defines T cell lineage, and CLL-1 is a myeloid lineage-restricted cell surface marker. Human platelet-derived growth factor receptor (PDGFR) is commonly used for the protein expression in mammalian cell lines. Here, we used the transmembrane (TM) domain of PDGFR as a fusion partner to display single-chain variable fragment (scFv) antibodies on the exosomal surface. To generate dual-targeting aCD3-aCLL-1 SMART-Exos, two single chain variable fragment (scFv) antibodies were encoded in single polypeptides, which were genetically linked to the PDGFR TM domain (Figure 4C). Encoding two individual scFvs into single polypeptides is based on the idea to avoid potential steric hindrance between two antibody scalfolds. Additionally, aCD3 scFv antibodies and aCLL-1 scFv antibodies were separately fused with PDGFR TM domain for generation of monoclonal exosomes as controls (Figure 4A and 4B). A hemagglutinin (HA) epitope tag was fused at the N-terminus of each fusion protein.

Expression and identification of SMART-Exos. Expression constructs were transfected with Expi293F cells and cultured in Expi293 medium. The expressed SMART-Exos were harvested and isolated by dilferential centrifugation. The yield of 30 mL transfection of SMART-Exos is approximately 100 mg, containing -6.69 x 10 ¹⁰ exosome particles.

Western blot analysis indicated that all scFv antibodies were expressed in exosomes (Figure 5A). Moreover, SMART-Exos showed expression of exosomal marker CD9, CD81, and CD63, similar to native exosomes (Figure 5B).

Characterization of SMART-Exos. The aCD3-aCLL-1 SMART-Exos were imaged by transmission electron microscopy (Figure 6). Quantification and size determination of aCD3- aCLL-1 SMART-Exos was assessed by nanoparticle tracking analysis (NTA), indicating a size distribution peaking at 73 nm in diameter (Figure 7), which was consistent with previous studies [29], [40], [49]

Antigen expression analyses of AML cell lines. To analyze CLL-1 antigen expression levels on different AML cell lines (U937, HL60, and KG-1A), equal number of cells (500,000 cells/sample) were incubated with 20 nM FITC-conjugated aCLLl antibodies in PBS with 2% FBS on ice for 45 min. Cells were washed and analyzed by flow cytometry. U937 has the highest expression levels of CLL-1, followed by HL60. KG-1A has the lowest expression levels of CLL-1. (Figure 8)

Binding assays. Flow cytometric analysis indicated that aCD3-aCLL-1 SMART-Exos have significant binding affinity to CLL-1 positive cells and Jurkat cells, showing that scFv antibodies displayed on exosomal surface allow SMART-Exos to target both CLL-1 and CD3- expressing cells (Figure 9A, 9B, and 9C). For monospecific SMART-Exos, they exhibited selected binding to respective target cells. aCD3 SMART-Exos can only bind to Jurkat cells (Figure 9A), while aCLL-1 SMART-Exos only showed high binding affinity to both U937 and HL60 cells (Figure 9B, Figure 9C). None of the SMART-Exos displayed strong binding to KG-1A cells (CD3-, CLL-1-) (Figure 9D).

In vitro cytotoxicity assays. To determine whether the cytotoxicity of the SMART- Exos is associated with antigen abundance on target cells, three AML cell lines with various CLL1 expression levels, including U937 (CLL-1⁺⁺⁺), HL60 (CLL-1⁺⁺), and KG-1A (CLL-1⁺) were used to compare the cytotoxicities of SMART-Exos after an incubation period of 24 hours.

In the presence of human PBMCs (at an E:T ratio of 1:10), aCD3-aCLL-1 SMART- Exos exhibited highly potent and specific cytotoxicity against U937 cells with an EC₅₀ of 14.21 ± 1.10 ng/mL, followed by HL60 with an EC₅₀ of 82.84 ± 1.17 ng/mL and significantly decreased cytotoxicity for KG-1A cells with an EC₅₀ of 685.0 ± 1.31 ng/mL (Figure 10A, 10B, 10C). The cytotoxicity induced by SMART-Exos to target AML cell lines were positively correlated with levels of CLL-1 expression (Figure 11).

Our results showed that aCD3-aCLL-1 SMART-Exos possess remarkable potency and specificity towards CLL-1 -positive cells by redirecting T cells to induce immune attacks to target cells in an antigen-dependent manner, underscoring the promising potential of SMART- Exos as immunotherapeutics for AML.

Example 3. Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of a composition of a formula described herein, a composition specifically disclosed herein, or a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as 'Composition X'):

These formulations may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Composition X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest.

Example 4. Sequences

SEQ ID NOS 1-6: PRIM ER SEQU ENCES (listed in Table 1).

SEQ ID NO 9 is the amino acid sequence of the PDGFR TM domain:

SEQ ID NO 15 is HA-CLL1 scFv-linker-CD3 scFv-linker-PDGRF TM (the scFv portions in the opposite orientation of 1):

Sequences of the VL and Vh of the recited antibody orientations:



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.

All publications, patents, and patent documents are incorporated by reference herein, 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, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

What is claimed is:

1. An engineered extracellular vesicle comprising:

a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) an epitope tag, (b) a first antibody moiety, (c) a first peptide linker, (d) a second antibody moiety, (e) a second peptide linker, and (f) an extracellular vesicle membrane protein or portions thereof, wherein the fusion protein is displayed on a surface of the engineered extracellular vesicle.

2. The engineered extracellular vesicle of claim 1 wherein the extracellular vesicle comprises one or more of an exosome, a liposome, a micro vesicle, and an apoptotic body, each of the extracellular vesicle isolated from an eukaryotic cell or a prokaryotic cell.

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

4. The engineered extracellular vesicle of claim 1 wherein the first antibody moiety binds to a T-cell marker protein or a cancer cell surface-marker protein.

5. The engineered extracellular vesicle of claim 1 wherein the second antibody moiety binds to a T-cell marker protein or a cancer cell surface-marker protein.

6. The engineered extracellular vesicle of claim 4 wherein the T-cell or immune cell marker comprises one or more of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD24, CD25, CD27, CD28, CD30, CD31, CD38, CD40L, CD45, CD56, CD68, CD91, CD114, CD163, CD206, LFA1, PD1, ICOS, BTLA, KIR, CD137, OX40, LAG3, CTLA4, TIM3, B7-H3, B7-H4, and a T-cell Receptor.

7. The engineered extracellular vesicle of claim 5 wherein the cancer cell surface-marker is a cancer cell marker overexpressed by acute myeloid leukemia (AML) cancer cells.

8. The engineered extracellular vesicle of claim 7 wherein the cancer cell surface-marker comprises one or more of CLL-1, HER2, HER3, EGER, CD33, CD34, CD38, CD123, TIM3, CD25, CD32, and CD96.

9. The engineered extracellular vesicle of claim 1 wherein extracellular vesicle membrane protein comprises Platelet Derived Growth Factor Receptor (PDGFR), Lamp2b, lactadherin C1C2 domain, CD13, CD9, or portions thereof.

10. The engineered extracellular vesicle of claim 9 wherein extracellular vesicle membrane protein comprises a transmembrane domain of the PDGFR.

11. The engineered extracellular vesicle of claim 1 wherein the first antibody moiety comprises a first VL domain, a first VH domain, and a third peptide linker therebetween; and the second antibody moiety comprises a second VL domain, a second VH domain, and a fourth peptide linker therebetween.

12. The engineered extracellular vesicle of claim 11 wherein the first peptide linker, the second peptide linker, the third peptide linker, and the fourth peptide linker comprise a (GGGS)n peptide (SEQ ID NO: 18) where n is an integer from 1 to 5 or a (GGGGS)n peptide (SEQ ID NO: 19) where n is an integer from 1 to 5.

13. The engineered extracellular vesicle of claim 1 wherein the epitope tag comprises hemagglutinin.

14. The engineered extracellular vesicle of claim 1 wherein the engineered extracellular vesicle has a particle size of about 25 nm to about 150 nm.

15. 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).

16. The engineered extracellular vesicle of claim 15 wherein the first antibody scFv binds to a T-cell marker protein or an immune cell marker protein, the T-cell marker protein or the immune cell marker protein comprising CD3; and the second antibody scFv binds to a cancer cell surface-marker protein, the cell surface marker protein comprising CLL- 1.

17. The engineered extracellular vesicle of claim 15 wherein the first antibody scFv binds to a cancer cell surface marker protein, the cell surface marker protein comprising CLL-1; and the second antibody scFv binds to a T-cell marker protein or immune cell marker protein, the T-cell marker protein or immune cell marker protein comprising CD3.

18. The engineered extracellular vesicle of claim 16 wherein each of the first antibody scFv and the second antibody scFv comprise a VH and a VL domain, and an orientation of the first antibody scFv and the second antibody scFv is V_{L- aCD3} -V_{H- aCD3} -V_L-aCLL-1-V_H-aCLL-1 ( aCD3- aCLL-1 - scFv), V_{H- aCD3} -V_{L- aCD3} -V_H-aCLL-1 -V_H-aCLL-1 (aCD3-aCLL-1 scFv), V_{H- aCD3} -V_{L- aCD3} - -V_H-aCLL-1 -V_L-aCLL-1 (aCD3-aCLL-1 scFv), or V_{L- aCD3} -V_{H- aCD3} -V_H-aCLL-1 -V_L-aCLL-1 (aCD3- aCLL-1 scFv).

19. The engineered extracellular vesicle of claim 17 wherein each of the first antibody scFv and the second antibody scFv comprise a VH and a VL domain, and an orientation of the first antibody scFv and the second antibody scFv is V_H-aCLL-1 -V_L-aCLL-1 - V_{L- aCD3} -V_{H- aCD3} ( -aCLL- 1-aCD3 scFv), V_H-aCLL-1 -V_L-aCLL-1 - V_{H- aCD3} -V_{L- aCD3} ( -aCLL-1 - aCD3 scFv), V_L-aCLL-1 -V_H- _aCLL-1 - V_{L- aCD3} -V_{H- aCD3} (aCLL-1 - aCD3 ScFv), or V_L-aCLL-1 -V_H-aCLL-11 - V_{H- aCD3} - V_{L- aCD3}

(aCLL-1 - aCD3 scFv).

20. The engineered extracellular vesicle of claim 16 wherein the epitope tag comprises hemagglutinin, and the extracellular vesicle membrane protein is a transmembrane domain of PDGFR.

21. A method to target cancer cells, the method comprising:

a) forming an engineered extracellular vesicle according to claim 1 ; and

b) contacting a subject comprising cancer cells and healthy cells with the engineered extracellular vesicle;

wherein the engineered extracellular vesicle selectively targets the cancer cells.

22. The method of claim 16, wherein the first antibody moiety binds to a T-cell marker protein, the T-cell marker protein comprising CD3; and the second antibody moiety binds to a cancer cell surface marker protein, the cell surface marker protein comprising CLL-1.

23. The method of claim 21 wherein the cancer cells are AML cancer cells.

24. The method of claim 21 wherein the engineered extracellular vesicle selectively targets AML cancer cells by binding to CLL-1 receptors in the cancer cells and recruiting T-cells to the cancer cells, wherein the T-cells selectively kill the cancer cells.

25. An engineered extracellular vesicle comprising:

a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) a hemagglutinin epitope tag, (b) a first antibody single-chain variable fragment (scFv) that binds to a cancer cell surface marker, the cancer cell surface marker comprising CLL-1, (c) a first peptide linker, (d) a second antibody scFv that binds to a T-cell surface marker, the T-cell surface marker comprising CD3, (e) a second peptide linker, and (f) an extracellular vesicle membrane protein comprising the transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR), wherein the fusion protein is displayed on a surface of the engineered extracellular vesicle.

26. An engineered extracellular vesicle comprising:

a fusion protein comprising consecutive amino acids, beginning from the an amino terminus of the fusion protein, corresponding to (a) an epitope tag, (b) an antibody moiety, (c) a peptide linker, and (d) an extracellular vesicle membrane protein or portions thereof, wherein the fusion protein is displayed on a surface of the engineered extracellular vesicle.

27. The engineered extracellular vesicle of claim 26 wherein the antibody moiety binds to a T-cell or immune cell marker comprising one or more of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD24, CD25, CD27, CD28, CD30, CD31, CD38, CD40L, CD45, CD56, CD68, CD91, CD114, CD163, CD206, LFA1, PD1, ICOS, BTLA, KIR, CD137, OX40, LAG3, CTLA4, TIM3, B7-H3, B7-H4, and a T-cell Receptor.

28. The engineered extracellular vesicle of claim 26 wherein the antibody moiety binds to a cancer cell surface-marker comprising one or more of CLL-1, HER2, HER3, EGFR, CD33, CD34, CD38, CD123, TIM3, CD25, CD32, and CD96.

29. The engineered extracellular vesicle of claim 26 wherein the antibody moiety comprises a single chain variable fragment (scFv), a single domain antibody, a bispecific antibody, and a multispecific antibody.

30. The engineered extracellular vesicle of claim 26 wherein extracellular vesicle membrane protein comprises Platelet Derived Growth Factor Receptor (PDGFR), Lamp2b, lactadherin C1C2 domain, CD13, CD9, or portions thereof.

31. The engineered extracellular vesicle of claim 26 wherein the epitope tag comprises hemagluttinin;

the antibody moiety comprises a single chain variable fragment (scFv) that binds to CLL-1; and

the extracellular membrane protein comprises a transmembrane domain of Platelet Derived Growth Factor Receptor (PDGFR).

32. The engineered extracellular vesicle of claim 26 wherein the epitope tag comprises hemagluttinin;

the antibody moiety comprises a single chain variable fragment (scFv) that binds to CD3; and